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HK1094210B - Template-fixed beta-hairpin peptidomimetics with cxcr4 antagonizing activity - Google Patents

Template-fixed beta-hairpin peptidomimetics with cxcr4 antagonizing activity Download PDF

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Publication number
HK1094210B
HK1094210B HK06114147.7A HK06114147A HK1094210B HK 1094210 B HK1094210 B HK 1094210B HK 06114147 A HK06114147 A HK 06114147A HK 1094210 B HK1094210 B HK 1094210B
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HK
Hong Kong
Prior art keywords
arg
lower alkyl
pro
amino acid
cys
Prior art date
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HK06114147.7A
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Chinese (zh)
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HK1094210A1 (en
Inventor
J．祖姆布鲁恩
S．J．德马克
R．姆克赫伊
K．莫赫勒
J．A．鲁宾逊
H．翰泽
B．罗马格诺里
S．络茨伍罗
J．W．沃里耶布罗德
F．戈姆博特
D．欧布雷驰特
C．鲁丁
Original Assignee
波利弗尔股份公司
苏黎世大学
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Priority claimed from PCT/EP2003/004640 external-priority patent/WO2004096839A1/en
Application filed by 波利弗尔股份公司, 苏黎世大学 filed Critical 波利弗尔股份公司
Publication of HK1094210A1 publication Critical patent/HK1094210A1/en
Publication of HK1094210B publication Critical patent/HK1094210B/en

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Description

Template-fixed beta-hairpin peptidomimetics having CXCR4 antagonistic activity
Technical Field
The present invention provides template-fixed β -hairpin peptidomimetics comprising a template-fixed chain of 12, 14 or 18 α -amino acid residues, wherein the residues, depending on their position in the chain, are Gly, NMeGly, Pro or Pip, or are of some type as defined below. These template-fixed β -hairpin mimetics have CXCR4 antagonistic activity. In addition, the present invention provides an efficient synthetic method according to which these compounds can be prepared in a parallel library format (parallel library-format) as required. These β -hairpin peptidomimetics show improved efficacy, bioavailability, half-life and most importantly a significant increase in the ratio between CXCR4 antagonistic activity of the mimetic and erythrocyte hemolysis and cytotoxicity.
Background
Available treatments for treatment of HIV infection to date have resulted in significant improvements in symptoms or recovery in infected people. Although highly active antiretroviral therapy (HAART-therapy) involving the combined use of reverse transcriptase/protease inhibitors has significantly improved the clinical treatment of AIDS or HIV infected individuals, there are several serious problems including multi-drug resistance, considerable side effects and high costs. Particularly needed are anti-HIV agents that block HIV infection early in the infection, such as at the time of viral entry.
It has recently been recognized that the chemokine receptors CCR5 and CXCR4, as well as the primary receptor CD4, are required for human immunodeficiency viruses to efficiently enter target cells (n.levy, engl.j.med., 335, 29, 1528-. Therefore, drugs capable of blocking CXCR4 chemokine receptors should prevent infection in healthy individuals or slow or stop virus development in infected patients (Science, 1997, 275, 1261-1264).
Among the different types of CXCR4 inhibitors (m.schwarz, t.n.c.wells, a.e.i.prudfoot, Receptors and Channels, 2001, 7, 417-428), an emerging species is a natural cationic peptide analogue derived from Polyphemusin II, which has an antiparallel β -sheet structure and a β -hairpin (β -hairpin) held by two disulfide bridges (h.nakashima, m.masuda, t.murakami, y.koyanagi, a.matsumoto, n.fujii, n.yamamoto, aniticrobobiAgents and chemioth.1992, 36, 1249-1255; h.tamimura, m.kumada, m.masuda, a.otaka, s.nakoshi, h.h.yakoshii, h.yakoshii, m.h.kushii, m.m.masokou, n.yamoki, n.yamoaku.32, waystimu.p.n.32, wo.e.n.waystimu, m.32, biophysi.p.p.p.p.p.n.p.p.p. 95/10534, bio-koyagi, m.t.t, n.t.
The synthesis of structural analogs and structural studies using Nuclear Magnetic Resonance (NMR) spectroscopy have shown that the cationic peptide adopts a well-defined β -hairpin conformation, due to the restriction of one or two disulfide bridges. (H.Tamamura, M.Sugioka, Y.Odagaki, A.Omagari, Y.Kahn, S.Oishi, H.Nakashima, N.Yamamoto, S.C.Peiper, N.Hamamaka, A.Otaka, N.Fujii, bioorg.Med.Chem.Lett.2001, 359- "362). These results indicate that this β -hairpin structure plays an important role in CXCR4 antagonistic activity.
Additional structural studies have also shown that antagonistic activity can also be affected by modulation of the amphipathic structure and pharmacophore (H.Tamamura, A.Omagarai, K.Hiramatsu,. K.Gotoh, T.Kanamoto, Y.xu, E.Kodama, M.Matsuoka, T.Hattori, N.Yamamoto, H.Nakashima, A.Otaka, N.Fujii, bioorg.Med.Chem.Lett.2001, 11, 1897-containing 1902; H.Tamamura, A.Omagarai, K.Hiramatsu, S.Oishi, H.Habashita, T.Kanamoto, K.Gotoh, N.Yamamoto, H.Nakashima, A.Otaka N.Jii, Bioorg.Futom.14110, Yanaoko, K.10, K.Gotoh, N.Yamato-10, Hakashima, H.10, Haemaokamu.10, Hayaokamu.10, K.10, Haemaokra, H.S.1897, H.S.S.S.S.S.S.S.S.S.S.A.S.S.S.S.10, H.M.S.S.S.S.S.S.S.S.A.S.S.A.S.S.A.S.S.S.S.S.S.A.A.A.A.A.A.A.A.A.A..
A key issue in the design of CXCR4 antagonistic peptides is selectivity. polyphemusin II derived analogs, although improved, still have cytotoxicity (k. matsuzaki, m. fukui, n. fujii, k. miyajima, biochim. biophysis. acta 1991, 259, 1070; a. otaka, h. tamamamura, y. terakawa, m. masuda, t.koide, t.murakami, h.nakashima, k.matsuzaki, k.miyajima, t.ibuka, m.waki, a.matsumoto, n.yamamoto, n.fujii biol.pharm.bull.1994, 17, 1669 and references cited above).
This cytotoxic activity essentially precludes its use in vivo and is a serious drawback in clinical applications. Before intravenous use is considered, general toxicity, protein binding activity in serum and protease stability become serious problems that must be adequately solved.
It has recently been disclosed that the CXCR4 receptor is not only involved in the entry of HIV but also in the chemotactic activity of Cancer cells such as breast Cancer metastasis or ovarian Cancer (a. muller, b. homey, h. soto, n.ge, d.catron, m.e.buchan, t.mc Clanahan, e.murphey, w.yuan, s.n.wagner, j.luis Barrera, a.mohar, e.verastegui, a.zlik, Nature 2001, 50, 410, j.m.hall, k.s.korach, molecular endocrinology, 2003, 1-47), non-hodgkin lymphoma (f.berthini, c.dell agala, p.manusco, c.rabasco, a.buelini, s.burlai, sabiri, sartori, R. Blocking this chemotactic activity (chemotactic activity) with CXCR4 inhibitors can prevent the migration of cancer cells.
This CXCR4 receptor is also implicated in tumor growth and proliferation. Activation of the CXCR4 receptor has been shown to be critical for the growth of malignant neurons and glia tumors and small cell lung tumors. In addition, systemic administration of the CXCR4 antagonist AMD3100 may inhibit growth of intracranial glioblastomas and medulloblastoma xenografts by increasing apoptosis and decreasing proliferation of tumor cells (Rubin JB, Kung AL, Klein RS, Chan JA, Sun Y, SchmidK, Kieran MW, luter AD, Segal RA. Proc Natl Acad Sci USA 2003100 (23): 13513-.
Chemokine stromal cell-derived factor-1 (CXCL12/SDF-1) and its receptor CXCR4 are implicated in the trafficking of B cells and hematopoietic progenitor cells. This CXCR4 receptor has been shown to play an important role in the release of stem cells from the bone marrow into the peripheral blood. This receptor is expressed, for example, on CD34+ cells and has been implicated in the process of migration and homing of CD34+ cells. This activity of the CXCR4 receptor is very important for efficient apheresis (apheresis) collection of peripheral blood stem cells. Autologous peripheral blood cell autologous transplantation will provide rapid and sustained hematopoietic recovery following high dose chemotherapy or radiotherapy in patients with hematologic malignancies and solid tumors. (WC. Liles et al, Blood 2003, 102, 2728-.
There is increasing evidence that the interaction between chemokines in general, and the chemoattractant CXCL 12/stromal cell derived factor-1 α in particular, and its receptor CXCR4 plays a key role in angiogenesis. Chemokines induce angiogenesis either directly by binding to their cognate receptors on endothelial cells or indirectly by promoting inflammatory cell infiltrates that deliver other angiogenic stimuli. A number of pro-inflammatory chemokines, including interleukin 8(IL-8), growth-regulating oncogenes, stromal cell derived factor 1(SDF-1), monocyte chemotactic protein 1(MCP-1), eotaxin 1 and I-309, have been shown to be useful as direct inducers of angiogenesis. (Chen X, Beutler JA, McCloud TG, Loehfelm A, Yang L, Dong HF, Chertov OY, Salcedo R, Oppenheim JJ, Howard 0M. Clin Cancer Res.20039 (8): 3115-.
It is well established that chemokines are involved in many inflammatory pathological processes and that some of them play a key role in the regulation of osteoclast development. Immunostaining of SDF-1(CXCL12) on synovial and bone tissue biopsies obtained from Rheumatoid Arthritis (RA) and Osteoarthritis (OA) samples has revealed a strong increase in expression levels under inflammatory conditions. (Grassi F, Cristino S, Toneguzzi S, Picentini A, Facchini A, Lisignoli G.J Cell physiol.2004; 199 (2): 244-.
Mediation of the process of recruitment of immune cells to the site of inflammation should be stopped by CXCR4 inhibitors.
Novel strategies are employed to stabilize the beta-hairpin conformation in cyclic backbone-turn peptidomimetics that exhibit high CXCR4 antagonistic activity, are useful for efficient reinjection collection of peripheral blood stem cells, and have anti-cancer and anti-inflammatory activity, among the compounds described below.
This involves grafting a cationic and hydrophobic hairpin sequence onto a template that functions to constrain the peptide loop backbone to a hairpin geometry. The rigidity of the hairpin can be further influenced by the introduction of disulfide bonds. Hairpin mimetics which bind to the template have been described in the literature (D, Obrecht, M.Altorfer, J.A.Robinson, adv.Med.chem.1999, 4, 1-68; J.A.Robinson, Syn.Lett.2000, 4, 429-441), but such molecules have not previously been evaluated for the development of CXCR4 antagonistic peptides. However, the ability to generate β -hairpin peptidomimetics by using combinatorial or parallel synthetic methods is now established (L.Jiang, K.Moehle, B.Dhanapaal, D.Obrecht, J.A.Robinson, Helv.Chim.acta.2000, 83, 3097-.
These methods allow the synthesis and screening of large hairpin mimetic libraries, which in turn significantly facilitates structure-activity studies and thus the development of new molecules with highly potent CXCR4 antagonistic activity or anti-cancer activity or anti-inflammatory activity and low hemolytic activity towards human erythrocytes.
The β -hairpin peptidomimetics obtained by the methods described herein are useful as anti-HIV agents, anti-cancer agents, as inhibitors of tumor growth or as apoptosis inducers, anti-metastatic agents, and anti-inflammatory agents or as agents capable of use in the reinjection collection of peripheral blood stem cells.
Disclosure of Invention
The beta-hairpin peptidomimetics of the present invention are compounds of the general formula:
wherein
Is a radical of one of the formulae
And
wherein
Is a residue of Gly or L-alpha-amino acid, wherein B is of the formula-NR20CH(R71) -or an enantiomer of one of the groups a1 to a69 as defined below;
is a radical of one of the formulae
And
R1is H; a lower alkyl group; or aryl-lower alkyl;
R2is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R3Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R4Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)p(CHR61)sCOOR57；-(CH2)p(CHR61)sCONR58R59；
-(CH2)p(CHR61)sPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R5Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R6Is H; an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R7Is an alkyl group; an alkenyl group; - (CH)2)q(CHR61)sOR55；-(CH2)q(CHR61)sNR33R34；
-(CH2)q(CHR61)sOCONR33R75；-(CH2)q(CHR61)sNR20CONR33R82；
-(CH2)r(CHR61)sCOOR57；-(CH2)r(CHR61)sCONR58R59；
-(CH2)r(CHR61)sPO(OR60)2；
-(CH2)r(CHR61)sSO2R62(ii) a Or- (CH)2)r(CHR61)sC6H4R8；
R8Is H; cl; f; CF (compact flash)3；NO2(ii) a A lower alkyl group; a lower alkenyl group; an aryl group; aryl-lower alkyl;
-(CH2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；-(CH2)o(CHR61)NR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sCOR64；
R9Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R10Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R11Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R12Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)r(CHR61)sCOOR57；
-(CH2)r(CHR61)sCONR58R59；-(CH2)r(CHR61)sPO(OR60)2；-(CH2)r(CHR61)sSO2R62(ii) a Or- (CH)2)r(CHR61)sC6H4R8；
R13Is an alkyl group; an alkenyl group; - (CH)2)q(CHR61)sOR55；-(CH2)q(CHR61)sSR56；
-(CH2)q(CHR61)sNR33R34；
-(CH2)q(CHR61)sOCONR33R75；-(CH2)q(CHR61)sNR20CONR33R82；
-(CH2)q(CHR61)sCOOR57；-(CH2)q(CHR61)sCONR58R59；
-(CH2)q(CHR61)sPO(OR60)2；
-(CH2)q(CHR61)sSO2R62(ii) a Or- (CH)2)q(CHR61)sC6H4R8；
R14Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)q(CHR61)sCOOR57；-(CH2)q(CHR61)sCONR58R59；
-(CH2)q(CHR61)sPO(OR60)2；.
-(CH2)q(CHR61)sSOR62(ii) a Or- (CH)2)q(CHR61)sC6H4R8；
R15Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R16Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R17Is an alkyl group; an alkenyl group; - (CH)2)q(CHR61)sOR55；-(CH2)q(CHR61)sSR56；
-(CH2)q(CHR61)sNR33R34；
-(CH2)q(CHR61)sOCONR33R75；-(CH2)q(CHR61)sNR20CONR33R82；
-(CH2)q(CHR61)sCOOR57；-(CH2)q(CHR61)sCONR58R59；
-(CH2)q(CHR61)sPO(OR60)2；
-(CH2)q(CHR61)sSO2R62(ii) a Or- (CH)2)q(CHR61)sC6H4R8；
R18Is an alkyl group; an alkenyl group; - (CH)2)p(CHR61)sOR55；-(CH2)p(CHR61)sSR56；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)p(CHR61)sCOOR57；-(CH2)p(CHR61)sCONR58R59；
-(CH2)p(CHR61)sPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R19Is a lower alkyl group; - (CH)2)p(CHR61)sOR55；-(CH2)p(CHR61)sSR56；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)p(CHR61)sCOOR57；-(CH2)p(CHR61)sCONR58R59；
-(CH2)p(CHR61)sPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8(ii) a Or
R18And R19Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-；
Or- (CH)2)2NR57(CH2)2-；
R20Is H; an alkyl group; an alkenyl group; or aryl-lower alkyl;
R21is H; an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R22Is H; an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R23Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R24Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R25Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R26Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8(ii) a Or
R25And R26Together may form: - (CH)2)2-6-；-(CH2)rO(CH2)r-；-(CH2)rS(CH2)r-; or
-(CH2)rNR57(CH2)r-；
R27Is H; an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sOCONR33R75；
-(CH2)o(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or-(CH2)o(CHR61)sC6H4R8；
R28Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)s-OR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R29Is an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R30Is H; an alkyl group; an alkenyl group; or aryl-lower alkyl;
R31is H; an alkyl group; an alkenyl group; - (CH)2)p(CHR61)sOR55；-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R32Is H; a lower alkyl group; or aryl-lower alkyl;
R33is H; alkyl radicalAn alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR34R63；
-(CH2)m(CHR61)sOCONR75R82；-(CH2)m(CHR61)sNR20CONR78R82；
-(CH2)o(CHR61)sCOR64；-(CH2)o(CHR61)s-CONR58R59，
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R34Is H; a lower alkyl group; aryl, or aryl-lower alkyl;
R33and R34Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R35Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)p(CHR61)sCOOR57；-(CH2)p(CHR61)sCONR58R59；
-(CH2)p(CHR61)sPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)p(CHR61)sC6H4R8；
R36Is H, alkyl; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)p(CHR61)sCOOR57；-(CH2)p(CHR61)sCONR58R59；
-(CH2)p(CHR61)sPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R37Is H; f; br; cl; NO2；CF3(ii) a A lower alkyl group; - (CH)2)p(CHR61)sOR55；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R38Is H; f; br; cl; NO2；CF3(ii) a An alkyl group; an alkenyl group; - (CH)2)p(CHR61)sOR55；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R39Is H; an alkyl group; an alkenyl group; or aryl-lower alkyl;
R40is H; an alkyl group; an alkenyl group; or aryl-lower alkyl;
R41is H; f; br; cl; NO2；CF3(ii) a An alkyl group; an alkenyl group; - (CH)2)p(CHR61)sOR55；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R42Is H; f; br; cl; NO2；CF3(ii) a An alkyl group; an alkenyl group; - (CH)2)p(CHR61)sOR55；
-(CH2)p(CHR61)sNR33R34；
-(CH2)p(CHR61)sOCONR33R75；-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R43Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)o(CHR61)sCONR58R59；
-(CH2)o(CHR61)sPO(OR60)2；
-(CH2)o(CHR61)sSO2R62(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R44Is an alkyl group; an alkenyl group; - (CH)2)r(CHR61)sOR55；-(CH2)r(CHR61)sSR56；
-(CH2)r(CHR61)sNR33R34；
-(CH2)r(CHR61)sOCONR33R75；-(CH2)r(CHR61)sNR20CONR33R82；
-(CH2)r(CHR61)sCOOR57；-(CH2)r(CHR61)sCONR58R59；
-(CH2)r(CHR61)sPO(OR60)2；
-(CH2)r(CHR61)sSO2R62(ii) a Or- (CH)2)r(CHR61)sC6H4R8；
R45Is H; an alkyl group; an alkenyl group; - (CH)2)o(CHR61)sOR55；-(CH2)o(CHR61)sSR56；
-(CH2)o(CHR61)sNR33R34；
-(CH2)o(CHR61)sOCONR33R75；-(CH2)o(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR57；-(CH2)s(CHR61)sCONR58R59；
-(CH2)s(CHR61)sPO(OR60)2；
-(CH2)s(CHR61)sSO2R62(ii) a Or- (CH)2)s(CHR61)sC6H4R8；
R46Is H; an alkyl group; an alkenyl group; or- (CH)2)o(CHR61)pC6H4R8；
R47Is H; an alkyl group; an alkenyl group; or- (CH)2)o(CHR61)sOR55；
R48Is H; a lower alkyl group; a lower alkenyl group; or aryl-lower alkyl;
R49is H; an alkyl group; an alkenyl group; - (CHR)61)sCOOR57；(CHR61)sCONR58R59；
(CHR61)sPO(OR60)2；-(CHR61)sSOR62(ii) a Or- (CHR)61)sC6H4R8；
R50Is H; a lower alkyl group; or aryl-lower alkyl;
R51is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)pPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)p(CHR61)sC6H4R8；
R52Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)pPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)p(CHR61)sC6H4R8；
R53Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sSR56；
-(CH2)m(CHR61)sNR33R34；-(CH2)m(CHR61)sOCONR33R75；
-(CH2)m(CHR61)sNR20CONR33R82；-(CH2)o(CHR61)sCOOR57；
-(CH2)o(CHR61)sCONR58R59；-(CH2)o(CHR61)pPO(OR60)2；
-(CH2)p(CHR61)sSO2R62(ii) a Or- (CH)2)p(CHR61)sC6H4R8；
R54Is H; an alkyl group; an alkenyl group; - (CH)2)m(CHR61)sOR55；-(CH2)m(CHR61)sNR33R34；
-(CH2)m(CHR61)sOCONR33R75；-(CH2)m(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)COOR57；-(CH2)o(CHR61)sCONR58R59(ii) a Or- (CH)2)o(CHR61)sC6H4R8；
R55Is H; a lower alkyl group; a lower alkenyl group; aryl-lower alkyl; - (CH)2)m(CHR61)sOR57；
-(CH2)m(CHR61)sNR34R63；-(CH2)m(CHR61)sOCONR75R82；
-(CH2)m(CHR61)sNR20CONR78R82；-(CH2)o(CHR61)s-COR64；
-(CH2)o(CHR61)COOR57(ii) a Or
-(CH2)o(CHR61)sCONR58R59；
R56Is H; a lower alkyl group; a lower alkenyl group; aryl-lower alkyl; - (CH)2)m(CHR61)sOR57；
-(CH2)m(CHR61)sNR34R63；-(CH2)m(CHR61)sOCONR75R82；
-(CH2)m(CHR61)sNR20CONR78R82；-(CH2)o(CHR61)s-COR64(ii) a Or
-(CH2)o(CHR61)sCONR58R59；
R57Is H; a lower alkyl group; a lower alkenyl group; aryl lower alkyl; orHeteroaryl lower alkyl;
R58is H; a lower alkyl group; a lower alkenyl group; an aryl group; a heteroaryl group; aryl-lower alkyl; or heteroaryl-lower alkyl;
R59is H; a lower alkyl group; a lower alkenyl group; an aryl group; a heteroaryl group; aryl-lower alkyl; or heteroaryl-lower alkyl; or
R58And R59Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R60Is H; a lower alkyl group; a lower alkenyl group; an aryl group; or aryl-lower alkyl;
R61is an alkyl group; an alkenyl group; an aryl group; a heteroaryl group; aryl-lower alkyl; heteroaryl-lower alkyl; - (CH)2)mOR55；
-(CH2)mNR33R34；-(CH2)mOCONR75R82；-(CH2)mNR20CONR78R82；-(CH2)oCOOR37；
-(CH2)oNR58R59(ii) a Or- (CH)2)oPO(COR60)2；
R62Is a lower alkyl group; a lower alkenyl group; aryl, heteroaryl; or aryl-lower alkyl;
R63is H; a lower alkyl group; a lower alkenyl group; aryl, heteroaryl; aryl-lower alkyl; heteroaryl-lower alkyl;
-COR64；-COOR57；-CONR58R59；-SO2R62(ii) a OR-PO (OR)60)2；
R34And R63Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R64Is H; a lower alkyl group; a lower alkenyl group; an aryl group; a heteroaryl group; aryl-lower alkyl; heteroaryl-lower alkyl;
-(CH2)p(CHR61)sOR65；-(CH2)p(CHR61)sSR66(ii) a Or- (CH)2)p(CHR61)sNR34R63；
-(CH2)p(CHR61)sOCONR75R82；-(CH2)p(CHR61)sNR20CONR78R82；
R65Is H; a lower alkyl group; a lower alkenyl group; aryl, aryl-lower alkyl; heteroaryl-lower alkyl; -COR57；-COOR57(ii) a or-CONR58R59；
R66Is H; a lower alkyl group; a lower alkenyl group; an aryl group; aryl-lower alkyl; heteroaryl-lower alkyl; or-CONR58R59；
m is 2 to 4; o is 0 to 4; p is 1 to 4; q is 0 to 2; r is 1 or 2; s is 0 or 1;
z is a chain of N alpha-amino acid residues, N being an integer 12, 14 or 18, wherein the positions of the amino acid residues in the chain are counted starting from the N-terminal amino acid, whereby these amino acid residues are Gly, NMeGly, Pro or Pip, or of the formula-A-CO-, or of the formula-B-CO-, depending on their position in the chain, or are of one of the following types:
C：-NR20CH(R72)CO-；
D：-NR20CH(R73)CO-；
E：-NR20CH(R74)CO-；
F：-NR20CH(R84) CO-; and
H：-NR20-CH(CO-)-(CH2)4-7-CH(CO-)-NR20-；
-NR20-CH(CO-)-(CH2)pSS(CH2)p-CH(CO-)-NR20-；
-NR20-CH(CO-)-(-(CH2)pNR20CO(CH2)p-CH(CO-)-NR20-; and
-NR20-CH(CO-)-(-(CH2)pNR20CONR20(CH2)p-CH(CO-)-NR20-；
I：-NR86CH2CO-；
R71is a lower alkyl group; a lower alkenyl group; - (CH)2)p(CHR61)sOR75；
-(CH2)p(CHR61)sSR75；
-(CH2)p(CHR61)sNR33R34；-(CH2)p(CHR61)sOCONR33R75；
-(CH2)p(CHR61)sNR20CONR33R82；
-(CH2)o(CHR61)sCOOR75；-(CH2)pCONR58R59；-(CH2)pPO(OR62)2；
-(CH2)pSO2R62(ii) a Or
-(CH2)o-C6R67R68R69R70R76；
R72Is H, lower alkyl; a lower alkenyl group; - (CH)2)p(CHR61)sOR85(ii) a Or
-(CH2)p(CHR61)sSR85；
R73Is- (CH)2)oR77；-(CH2)rO(CH2)oR77；-(CH2)rS(CH2)oR77(ii) a Or
-(CH2)rNR20(CH2)oR77；
R74Is- (CH)2)pNR78R79；-(CH2)pNR77R80；-(CH2)pC(＝NR80)NR78R79；
-(CH2)pC(＝NOR50)NR78R79；
-(CH2)pC(＝NNR78R79)NR78R79；-(CH2)pNR80C(＝NR80)NR78R79；
-(CH2)pN＝C(NR78R80)NR79R80；-(CH2)pC6H4NR78R79；-(CH2)pC6H4NR77R80；
-(CH2)pC6H4C(＝NR80)NR78R79；-(CH2)pC6H4C(＝NOR50)NR78R79；
-(CH2)pC6H4C(＝NNR78R79)NR78R79；-(CH2)pC6H4NR80C(＝NR80)NR78R79；
-(CH2)pC6H4N＝C(NR78R80)NR79R80；-(CH2)rO(CH2)mNR78R79；
-(CH2)rO(CH2)mNR77R80；
-(CH2)rO(CH2)pC(＝NR80)NR78R79；-(CH2)rO(CH2)pC(＝NOR50)NR78R79；
-(CH2)rO(CH2)pC(＝NNR78R79)NR78R79；-(CH2)rO(CH2)mNR80C(＝NR80)NR78R79；
-(CH2)rO(CH2)mN＝C(NR78R80)NR79R80；-(CH2)rO(CH2)pC6H4CNR78R79；
-(CH2)rO(CH2)pC6H4C(＝NR80)NR78R79；-(CH2)rO(CH2)pC6H4C(＝NOR50)NR78R79；
-(CH2)rO(CH2)pC6H4C(＝NNR78R79)NR78R79；
-(CH2)rO(CH2)pC6H4NR80C(＝NR80)NR78R79；-(CH2)rS(CH2)mNR78R79；
-(CH2)rS(CH2)mNR77R80；-(CH2)rS(CH2)pC(＝NR80)NR78R79；
-(CH2)rS(CH2)pC(＝NOR50)NR78R79；-(CH2)rS(CH2)pC(＝NNR78R79)NR78R79；
-(CH2)rS(CH2)mNR80C(＝NR80)NR78R79；-(CH2)rS(CH2)mN＝C(NR78R80)NR79R80；
-(CH2)rS(CH2)pC6H4CNR78R79；-(CH2)rS(CH2)pC6H4C(＝NR80)NR78R79；
-(CH2)rS(CH2)pC6H4C(＝NOR50)NR78R79；
-(CH2)rS(CH2)pC6H4C(＝NNR78R79)NR78R79；
-(CH2)rS(CH2)pC6H4NR80C(＝NR80)NR78R79；-(CH2)pNR80COR64；
-(CH2)pNR80COR77；
-(CH2)pNR80CONR78R79；-(CH2)pC6H4NR80CONR78R79(ii) a Or
-(CH2)pNR20CO-[(CH2)u-X]t-CH3Wherein X is-O-; -NR20-, or-S-; u is 1-3 and t is 1-6;
R75is a lower alkyl group; a lower alkenyl group; or aryl-lower alkyl;
R33and R75Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R75And R82Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R76Is H; a lower alkyl group; a lower alkenyl group; aryl-lower alkyl; - (CH)2)oOR72；
-(CH2)oSR72；
-(CH2)oNR33R34；-(CH2)oOCONR33R75；-(CH2)oNR20CONR33R82；
-(CH2)oCOOR75；-(CH2)oCONR58R59；-(CH2)oPO(OR60)2；-(CH2)pSO2R62(ii) a Or
-(CH2)oCOR64；
R77is-C6R67R68R69R70R76(ii) a Or heteroaryl having one of the following formulae
R78Is H; a lower alkyl group; an aryl group; or aryl-lower alkyl;
R78and R82Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-；
Or- (CH)2)2NR57(CH2)2-；
R79Is H; a lower alkyl group; an aryl group; or aryl-lower alkyl; or
R78And R79Together may be- (CH)2)2-7-；-(CH2)2O(CH2)2-; or- (CH)2)2NR57(CH2)2-；
R80Is H; or lower alkyl;
R81is H; a lower alkyl group; or aryl-lower alkyl;
R82is H; a lower alkyl group; an aryl group; a heteroaryl group; or aryl-lower alkyl;
R33and R82Together may form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-；
Or- (CH)2)2NR57(CH2)2-；
R83Is H; a lower alkyl group; an aryl group; or-NR78R79；
R84Is- (CH)2)m(CHR61)sOH；-(CH2)pCOOR80；-(CH2)m(CHR61)sSH；
-(CH2)pCONR78R79；-(CH2)pNR80CONR78R79；-(CH2)pC6H4CONR78R79(ii) a Or
-(CH2)pC6H4NR80CONR78R79；
R85Is a lower alkyl group; or lower alkenyl;
R86is R74；-(CH2)oR77；-(CH2)o-CHR33R75(ii) a Or
-[(CH2)u-X′]t-(CH2)vNR78R79(ii) a Or
-[(CH2)u-X′]t-(CH2)v-C(＝NR80)NR78R79Wherein X is-O-, -NR-20-，-S-；
or-OCOO-, u is 1-3, t is 1-6, and v is 1-3;
with the proviso that in the chain Z of n alpha-amino acid residues
-if n is 12, the amino acid residues in positions 1 to 12 are:
-P1: (ii) is of type C or type D or type E or type F, or the residue is Pro or Pip;
-P2: belonging to type E, or type F or the residue is Gly, NMeGly, Pro or Pip;
-P3: belonging to type E, or type F;
-P4: belonging to type C, or type D, or type F, or the residue is Gly or NMeGly;
-P5: belong to type E, or type D, or type C, or type F, or have the general formula-A-CO-or the residue is Gly, NMeGly, Pro or Pip;
-P6: belonging to type E, or type F, or having the general formula-B-CO-, or the residue is Gly or NMeGly;
-P7: belonging to type C, or type E or type F;
-P8: is of type D, or type C, or the residue is Pro or Pip;
-P9: belonging to type C, or type D or type F, or the residue is Gly or NMeGly;
-P10: is of type D, or type C, or the residue is Pro or Pip;
-P11: (ii) is of type E or type F or the residue is Gly or NMeGly; or
-P12: (ii) is of type C or type D or type E or type F, or the residue is Pro or Pip; or
-P4 and P9 and/or P2 and P11 together are capable of forming a group of type H;
d-isomers at P4, P6, P9 are also possible; and
-if n is 14, the amino acid residues in positions 1 to 14 are:
-P1: belonging to type C, or type D, or type E, or type F, or the residue is Gly or NMeGly or Pro or Pip;
-P2: belonging to type E, or type F, or type I, or type D;
-P3: belonging to type E, or type F; or type D, or type C, or the residue is Gly, NMeGly, Pro or Pip;
-P4: belonging to type D, or type C, or type F, or type E;
-P5: belonging to type E, or type F, or type C or type I;
-P6: belonging to type C, or type D, or type F, or the residue is Gly, NMeGly, Pro or Pip;
-P7: belonging to type C, or type D, or having the general formula-A-CO-, or the residue is Gly, NMeGly, Pro or Pip;
-P8: belonging to type E, or type F, or having the general formula B-CO-or belonging to type I or type D, or the residue is Pro or Pip;
-P9: belonging to type F, or type E, or type I, or type D, or the residue is Pro or Pip;
-P10: belonging to type F, or type D, or type C;
-P11: belonging to type D, or type C, or type F, or type E, or the residue is Pro or Pip;
-P12: belonging to type C, or type D, or type E, or type F;
-P13: belonging to type F, or type E, or the residue is Gly, NMeGly, Pro or Pip; and is
-P14: belonging to type F or type E or type C; or
-P2 and P13 and/or P4 and P11 together may form a group of type H;
d-isomers at P4, P7, P8 and P11 are possible;
further preconditions are
-the amino acid residue at position P1 is Gly or NMeGly or Pip; and/or
-the amino acid residue at position P2 belongs to type F or type I; and/or
-the amino acid residue at position P3 belongs to type F, or it is Gly, NMeGly, Pro or Pip; and/or
-the amino acid residue at position P4 belongs to type F; and/or
-the amino acid residue at position P5 belongs to type C or type F or type I; and/or
-the amino acid residue at position P6 belongs to type C or type D, or it is Gly or NMeGly; and/or
-the amino acid residue at position P7 belongs to type C or type D, or it is Pro, Pip or NMeGly; and/or
-the amino acid residue at position P8 is of type I or type D, or it is Pro or Pip; and/or
-the amino acid residue at position P9 belongs to type F or type I, or it is Pip; and/or
-the amino acid residue at position P10 belongs to type F; and/or
-the amino acid residue at position P11 belongs to type C, or it is Pip; and/or
-the amino acid residue at position P12 belongs to type C or type F; and/or
-the amino acid residue at position P13 belongs to type F, or it is Gly, NMeGly or Pip; and/or
-P2 forms together with P13 a group of type H; and/or
-P4 forms together with P11 a group of type H; and/or
-the amino acid residue at position P4 is the D-isomer; and/or
-the amino acid residue at position P11 is the D-isomer; and
-if n is 18, the amino acid residues in positions 1 to 18 are:
-P1: belonging to type D, or type E, or type C, or type F;
-P2: belonging to type E, or type F, or type D;
-P3: is of type C, or type D;
-P4: belonging to type E, or type D, or type F;
-P5: belonging to type D, or type C, or type E;
-P6: belonging to type C, or type E, or type F;
-P7: belonging to type C, or type D, or type E, or type F;
-P8: belonging to type F, or type E, or the residue is Gly or NMeGly;
-P9: belonging to type C, or type D, or type F;
-P10: is of type C, or of type E, or has the general formula-A-CO-, or the residue is Pro or Pip;
-P11: belonging to type C, or type E, or having the general formula-B-CO-, or the residue is Gly, NMeGly, Pro or Pip;
-P12: belonging to type D, or type C, or type F;
-P13: belonging to type E, or type F, or the residue is Gly or NMeGly;
-P14: belonging to type C, or type D, or type F;
-P15: belonging to type E, or type F;
-P16: belonging to type D, or type E, or type F;
-P17: belonging to type E, or type F; and is
-P18: belonging to type C, or type D, or type E, or type F; or
-P4 and P17 and/or P6 and P15 and/or P8 and P13 together are able to form a group of type H;
d-isomers at P10, P11 and P12 are also possible.
According to the invention, these beta-hairpin peptidomimetics can be prepared by a method comprising
(a) Coupling a suitably functionalized solid support with a suitably N-protected derivative of an amino acid, wherein the amino acid is at position 5, 6 or 7 in the desired end product if N is 12, or at position 6, 7 or 8 in the desired end product if N is 14, or at position 8, 9 or 10 in the desired end product if N is 18, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected;
(b) removing the N-protecting group from the product obtained;
(c) coupling the obtained product with a suitably N-protected derivative of an amino acid located in the desired end product at a position closer to the N-terminal amino acid residue, any functional groups that may be present in said N-protected amino acid derivative being likewise suitably protected;
(d) removing the N-protecting group from the product obtained;
(e) repeating steps (c) and (d) until the N-terminal amino acid residue is introduced;
(f) coupling the product obtained with a compound of the general formula
Wherein
As defined above and X is an N-protecting group or, additionally, if
The above group (a1) or (a2), then
(fa) coupling the product obtained in step (e) with an appropriately N-protected derivative of an amino acid of the general formula
HOOC-B-H III or HOOC-A-H IV
Wherein B and A are as defined above, any functional groups that may be present in the N-protected amino acid derivative are likewise suitably protected;
(fb) removing the N-protecting group from the obtained product; and
(fc) coupling the product obtained with a suitably N-protected derivative of an amino acid of formula IV or formula III above, respectively, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected; or, if
Is the above group (a3), then
(fa') coupling the product obtained in step (e) with a suitably N-protected derivative of an amino acid of formula III above, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected;
(fb') removing the N-protecting group from the obtained product; and
(fc') coupling the product obtained with a suitably N-protected derivative of an amino acid of formula III above, any functional groups which may be present in the N-protected amino acid derivative being likewise suitably protected;
(g) removing the N-protecting group from the product obtained in step (f) or (fc');
(h) coupling the product obtained with an appropriately N-protected derivative of an amino acid which is located at position 12 if N is 12, or at position 14 if N is 14, or at position 18 if N is 18 in the desired end product, any functional groups which may be present in said N-protected amino acid derivative being likewise appropriately protected;
(i) removing the N-protecting group from the product obtained;
(j) coupling the product obtained with a suitably N-protected derivative of an amino acid which is located at a position further away from position 12 if N is 12, or at a position further away from position 14 if N is 14, or at a position further away from position 18 if N is 18 in the desired end product, any functional groups which may be present in the N-protected amino acid derivative being likewise suitably protected;
(k) removing the N-protecting group from the product obtained;
(1) repeating steps (j) and (k) until all amino acid residues are introduced;
(m) if desired, selectively deprotecting one or more protected functional groups present in the molecule and appropriately replacing the released reactive groups;
(n) if desired, forming one, two or three interchain bonds between the side chains of suitable amino acid residues located at opposite positions of the β -strand region;
(o) detaching the obtained product from the solid support;
(p) cyclizing the product cleaved from the solid support;
(q) removing any protecting groups present on the functional groups of any members of the chain of amino acid residues and, if desired, any protecting groups that may otherwise be present in the molecule; and
(r) if desired, converting the obtained product into a pharmaceutically acceptable salt or converting the obtained pharmaceutically acceptable or unacceptable salt into the corresponding free compound of formula I or into a different pharmaceutically acceptable salt.
Alternatively, the peptidomimetics of the invention can be prepared in the following manner
(a') coupling a suitably functionalized solid support with a compound of the general formula
Wherein
As defined above and X is an N-protecting group or, additionally, if
The above group (a1) or (a2), then
(a' a) coupling of a suitably functionalized solid support with a suitably N-protected derivative of an amino acid of the general formula
HOOC-B-H III or HOOC-A-H IV
Wherein B and A are as defined above, any functional groups which may be present in the N-protected amino acid derivative are likewise suitably protected;
(a' b) removing the N-protecting group from the obtained product; and
(a' c) coupling the product obtained with a suitably N-protected derivative of an amino acid of formula IV or formula III above, respectively, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected; or if
Is the above group (a3), then
(a 'a') coupling the product obtained in step (e) with a suitably N-protected derivative of an amino acid of formula III above, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected;
(a 'b') removing the N-protecting group from the obtained product; and
(a 'c') coupling the product obtained with a suitably N-protected derivative of an amino acid of formula III above, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected;
(b ') removing the N-protecting group from the product obtained in step (a '), (a ' c) or (a ' c ');
(c') coupling the product obtained with a suitably N-protected derivative of an amino acid, wherein the amino acid is located at position 12 if N is 12, or at position 14 if N is 14, or at position 18 if N is 18 in the desired end product, any functional groups which may be present in the N-protected amino acid derivative being likewise suitably protected;
(d') removing the N-protecting group from the product obtained;
(e') coupling the product obtained with a suitably N-protected derivative of an amino acid located at a position further away from position 12 if N is 12, or at a position further away from position 14 if N is 14, or at a position further away from position 18 if N is 18 in the desired end product, any functional group which may be present in said N-protected amino acid derivative being likewise suitably protected;
(f') removing the N-protecting group from the product obtained;
(g ') repeating steps (e ') and (f ') until all amino acid residues have been introduced;
(h') if desired, selectively deprotecting one or several protected functional groups present in the molecule and appropriately replacing the released reactive groups;
(i') if desired, forming one, two or three interchain bonds between the side chains of suitable amino acid residues located at opposite positions of the β -strand region;
(j') separating the product obtained from the solid support;
(k') cyclizing the product cleaved from the solid support;
(l') removing any protecting groups present on the functional groups of any member of the chain of amino acid residues and, if desired, any protecting groups that may otherwise be present in the molecule; and
(m') if desired, converting the obtained product into a pharmaceutically acceptable salt or converting the obtained pharmaceutically acceptable or unacceptable salt into the corresponding free compound of the general formula I or into a different pharmaceutically acceptable salt.
The introduction of amino acid residues of type I can additionally be carried out by coupling with acylating agents containing a leaving group, such as bromine, chlorine or iodoacetic acid, and subsequently with the general formula H2N-R86Is subjected to nucleophilic displacement, wherein the amine is suitably protected if necessary.
The peptidomimetics of the invention may also be enantiomers of the compounds of formula I. These enantiomers can be prepared by modifications of the above process, wherein all enantiomers of chiral starting materials are used.
The term "alkyl" as used in the present specification, alone or in combination, denotes a saturated, straight or branched chain hydrocarbon radical having up to 24, preferably up to 12, carbon atoms. Similarly, the term "alkenyl" denotes a straight or branched chain hydrocarbon radical having up to 24, preferably up to 12 carbon atoms and containing at least one or (depending on the chain length) up to four olefinic double bonds. The term "lower" denotes groups and compounds having up to 6 carbon atoms. Thus, for example, the term "lower alkyl" denotes a saturated, straight-chain or branched-chain hydrocarbon group having up to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and the like. The term "aryl" denotes an aromatic carbocyclic hydrocarbon radical containing one or two six-membered rings, such as phenyl or naphthyl, which may be substituted by up to three substituents such as Br, Cl, F, CF3，NO2Lower alkyl or lower alkenyl. The term "heteroaryl" denotes an aromatic heterocyclyl group containing one or two five-and/or six-membered rings, wherein at least one of the rings contains up to three heteroatoms selected from O, S and N and the ring is optionally substituted; representative examples of such optionally substituted heteroaryl groups are as described above in connection with R77The definition of (a) is shown.
The structural element-A-CO-represents an amino acid structural unit, which is combined with the structural element-B-CO-to form the templates (a1) and (a 2). The structural element-B-CO-is combined with another structural element-B-CO-to form a template (a 3). Preferably, the template (a3) is only present in formula I, wherein n is 18 in the chain Z. The templates (a) to (p) constitute a structural unit whose N-and C-terminals are spatially oriented such that the distance between the two groups can be 4.0 to 5.5 angstroms. The peptide chain Z is linked to the C-and N-termini of the templates (a) to (p) via the respective N-and C-termini, such that the template and the chain form a cyclic structure, such as the one described in general formula I. For the case where the distance between the N-and C-termini of the template is between 4.0-5.5 angstroms, the template will induce the H-bond network required for the formation of the beta-hairpin conformation in peptide chain Z. Thus, the template and peptide chain form a β -hairpin mimetic.
This beta-hairpin conformation is highly correlated with CXCR4 antagonistic activity of the beta-hairpin mimetics of the invention. The β -hairpin stable conformational nature of the templates (a) to (p) plays a key role not only for the selective CXCR4 antagonistic activity but also for the synthetic methods defined above, since the introduction of the template at the beginning or near the middle of the linear protected peptide precursor significantly increases the cyclization yield.
The structural units A1-A69 belong to the class of amino acids in which the N-terminus is a secondary amine which forms part of a ring. Among the genetically encoded amino acids, only proline belongs to this class. The structural units A1 to A69 are in the configuration (D) and they are bound to the structural unit-B-CO-in the (L) -configuration. Preferred binding for the template (a1) isDAl-CO-LB-CO-toDA69-CO-LB-CO-. Thus, for example,DPro-Lpro constitutes a prototype of template (a 1). Less preferred, but possible, is the formation of a conjugate of the template (a2)LAl-CO-DB-CO-toLA69-CO-DB-CO-. Thus, for example,LPro-Dpro constitutes a prototype of template (a 2).
It will be appreciated that the structural unit in which A has the (D) -configuration-A1-CO-to-A69-CO-carries the group R in the alpha-position to the N-terminus1。R1Are H and lower alkyl, wherein R is1The most preferred values of (b) are H and methyl. Those skilled in the art will recognize that A1-A69 is shown in the (D) -configuration for R1Is the case for H and methyl, which corresponds to the (R) -configuration. Dependent on R1According to Cahn, Ingold or Prelog-law, this configuration may also be represented as (S).
Except for R1In addition, the structural units-A1-CO-to-A69-CO-can also carry a group denoted R2To R17Other substituents of (1). These other substituents may be H, and if it is not H, it is preferably a small to medium size aliphatic or aromatic group. R2To R17Examples of preferred values of (1)The method comprises the following steps:
-R2: h; a lower alkyl group; a lower alkenyl group; (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; r33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；R57: h; or lower alkyl); (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form- (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R3: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R4: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or is low inAlkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R5(ii) a A lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)oNR20CONR33R88(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r54: an alkyl group; an alkenyl group; an aryl group; aryl-lower alkyl; heteroaryl-lower alkyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R6: h; a lower alkyl group; low gradeAn alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R7: a lower alkyl group; a lower alkenyl group; - (CH)2)qOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)qSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)qNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)qNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)rCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)qCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)rPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); (CH)2)rSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)3NR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R9: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOO57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R10: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl)；-(CH2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R11: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R12: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)rCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)rCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)rPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R13: a lower alkyl group; a lower alkenyl group; - (CH)2)qOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)qSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)qNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)rCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)qCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)rPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)rSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R14: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form- (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; a lower alkyl group; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); - (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R15: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lowA lower alkyl group; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); particularly preferred is NR20CO lower alkyl (R)20H; or lower alkyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R16: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R17: a lower alkyl group; a lower alkenyl group; - (CH)2)qOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)qSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)qNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)qN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)rCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)qCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)rPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)rSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
Among the structural units A1 to A69, the following are preferred: r2A5, A8, A22, A25, R for H2A38, A42, A47 and A50 for H. Most preferred are structural units of type A8':
wherein R is20Is H or lower alkyl; and R is64Is an alkyl group; an alkenyl group; an aryl group; aryl-lower alkyl; or heteroaryl-lower alkyl; especially wherein R64Is n-hexyl (A8' -1); n-heptyl (A8' -2); 4- (phenyl) benzyl (A8' -3); benzhydryl (A8' -4); 3-amino-propyl (A8' -5); 5-amino-pentyl (A8' -6); methyl (A8' -7); ethyl (A8' -8); isopropyl (A8' -9); isobutyl (A8' -10); n-propyl (A8' -11); cyclohexyl (A8' -12); cyclohexylmethyl (A8' -13); n-butyl (A8' -14); phenyl (A8' -15); benzyl (A8' -16); (3-indolyl) methyl (A8' -17); 2- (3-indolyl) ethyl (A8' -18); (4-phenyl) phenyl (A8' -19); and n-nonyl (A8' -20).
The structural unit A70 belongs to the open-chain alpha-substituted alpha-amino acids, the structural units A71 and A72 belong to the corresponding beta-amino acid analogues, and the structural units A73-A104 belong to the cyclic analogues of A70. Such amino acid derivatives have been shown to restrict small peptides to well-established turn or U-shaped conformations (C.M. Venkatachalam, Biopolymers, 1968, 6, 1425-one 1434; W.Kabsch, C Sander, Biopolymers 1983, 22, 2577). Such Building Blocks or templates are ideally suited for stabilizing the beta-hairpin conformation in the Peptide loop (D.Obrecht, M.Altorfer, J.A.Robinson, "Novel Peptide mapping Blocks and templates for Efficient Lead filing", adv.Med chem.1999, Vol.4, 1-68; P.Balaram, "Non-standing amino acids in Peptide design and Peptide engineering", Current.Opin.Struct.biol.1992, 2, 845; M.Crisma, G.Valle, C.Toniolo, S.Prasa, R.B.Rao, P.Balartu, ". beta-nucleotides in molecules, structures of S.Prasid, R.B.Rao, P.Balartu,". beta-hairpin binding molecules, molecular binding of S.Prasid.262, J.1990, Al.J.J.249, J.J.Biocoding. J.J.Pat.J.35, J.J.249, J.J.Pat.J.35, J.J.J.J..
It has been shown that the enantiomers of the building Blocks-A70-CO-to A104-CO-in combination with the L-configured building block-B-CO-are both effective in stabilizing and inducing beta-hairpin conformations (D.Obrecht, M.Altorfer, J.A.Robinson, "Novel Peptide MimeticBuilding Blocks and Stredgees for effective Lead binding", Adv.Med chem.1999, vol.4, 1-68; D.Obrecht, C.Spiegler, P.Sch. nholzer, K.M uller, H.Heimgartner, F.Stierli, Helv.Chim.acta 1992, 75, 6-1696; D.Obrechhol, U.Dazel, J.Dal, C.Acronn, Buv. Chim.acta.1992, 75, 6-1696; D.Obrechhol, U.E.Schhuddalzehnsen, J.J.J.E.C.Schnei. Acronn.D.D.7. C.7. C.35, C.C.D.E.C.D.C.Pat.7, C.35, C.C.D.C.35, C.C.H.Pat., C.7, C.35, C.C.D.H.H.D.D.C.D.C.16632, C.E.C.C.C.E.C.C.C.S. Pat. No. Pat. 3, H.3, H.C.3, H.C.C.C.C.C.3, H.C.3, H.S. Pat. No. Pat. 4, H. No. Pat. No. 4, H.7, H. Pat. 4, H.C.C.35, H.35, H. No.3, H.7, C.C.5, H.35.
For the purposes of the present invention, template (A1) may therefore also consist of-A70-CO-to-A104-CO-in combination with the structural element-B-CO-in the (L) -configuration, where the structural elements A70 to A104 are of the (D) -or (L) -configuration.
In A70 to A104R20Preferred values of (b) are H or lower alkyl, with methyl being most preferred. R in the structural units A70 to A10418，R19And R21-R29Preferred values of (a) are the following:
-R18: a lower alkyl group.
-R19: a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)pCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)pCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)pSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)oC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R21: h; a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R22: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8: h; f; cl; CF; a lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R23: h; a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); particularly preferred is NR20CO lower alkyl (R)20H; or lower alkyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy);
-R24: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or (a)CH2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); particularly preferred is NR20CO lower alkyl (R)20H; or lower alkyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy);
-R25: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2(ii) a Wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R26: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(whereinR33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-or, R25And R26Together may be- (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or a lower alkyl group.
-R27: h; a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R28: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; orLower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R29: a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); particularly preferred is NR20CO lower-alkyl (R)20H; or lower alkyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
For templates (b) through (p), such as (b1) and (c1), preferred values for the respective symbols are the following:
-R8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; or lower alkyl; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R20: h; or a lower alkyl group.
-R30: h, methyl.
-R31: h; a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r32: h; or lower alkyl; or R33And R32Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); (-CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)rC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy); most preferred is-CH2CONR58R59(R58: h; or lower alkyl; r59: a lower alkyl group; or lower alkenyl).
-R32: h, methyl.
-R33: a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR34R63(wherein R is34: a lower alkyl group; or lower alkenyl; r63: h; or lower alkyl; or R34And R63Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)mOCONR75R82(wherein R is75: a lower alkyl group; or lower alkenyl; r82: h; or lower alkyl; or R75And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR78R82(wherein R is20: h; or lower alkyl; r78: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R78And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; low gradeAn alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl).
-R34: h; or a lower alkyl group.
-R35: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl).
-R36: a lower alkyl group; a lower alkenyl group; or aryl-lower alkyl.
-R37: h; a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R38: h; a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R78Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; lower alkylA group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R39: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl).
-R40: a lower alkyl group; a lower alkenyl group; or aryl-lower alkyl.
-R41: h; a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: is low inA lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form:-(CH2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R42: h; a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lowA lower alkyl group; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: lower alkyl, or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R43: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; orA lower alkyl group; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)xOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oPO(OR60)2(wherein R is60: a lower alkyl group; or lower alkenyl); - (CH)2)oSO2R62(wherein R is62: a lower alkyl group; or lower alkenyl); or- (CH)2)qC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R44: a lower alkyl group; a lower alkenyl group; - (CH)2)pOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)pSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)pNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R78Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)pN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)pCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)pCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)oC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R45: h; a lower alkyl group; a lower alkenyl group; - (CH)2)oOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)oSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)oNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)sOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)oN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)sC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R46: h; a lower alkyl group; a lower alkenyl group; - (CH)2)sOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)sSR56(wherein R is56: a lower alkyl group; or lower alkenyl); - (CH)2)sNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)sOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)sNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)sN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)sC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R47: h; OR OR55(wherein R is55: a lower alkyl group; or lower alkenyl).
-R48: h; or a lower alkyl group.
-R49: h; a lower alkyl group; - (CH)2)oCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)oCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or (CH)2)sC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R50: h; a methyl group.
-R51: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)pCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)pCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; is low inA lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)rC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R52: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-；R57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)pCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)pCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)rC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R53: h; a lower alkyl group; a lower alkenyl group; - (CH)2)mOR55(wherein R is55: a lower alkyl group; or lower alkenyl); - (CH)2)mNR33R34(wherein R is33: a lower alkyl group; or lower alkenyl; r34: h; or lower alkyl; or R33And R34Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mOCONR33R75(wherein R is33: h; or lower alkyl; or lower alkenyl; r75: a lower alkyl group; or R33And R75Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mNR20CONR33R82(wherein R is20: h; or lower alkyl; r33: h; or lower alkyl; or lower alkenyl; r82: h; or lower alkyl; or R33And R82Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); - (CH)2)mN(R20)COR64(wherein: R20: h; or lower alkyl; r64: a lower alkyl group; or lower alkenyl); - (CH)2)pCOOR57(wherein R is57: a lower alkyl group; or lower alkenyl); - (CH)2)pCONR58R59(wherein R is58: a lower alkyl group; or lower alkenyl; and R is59: h; a lower alkyl group; or R58And R59Together form: - (CH)2)2-6-；-(CH2)2O(CH2)2-；-(CH2)2S(CH2)2-; or- (CH)2)2NR57(CH2)2-; wherein R is57: h; or lower alkyl); or- (CH)2)rC6H4R8(wherein R is8：H；F；Cl；CF3(ii) a A lower alkyl group; a lower alkenyl group; or lower alkoxy).
-R54: a lower alkyl group; a lower alkenyl group; or aryl-lower alkyl.
Among the structural units A70 to A104 the following are preferredThe following steps: r22A74, A75, A76, R for H22A77, a78 and a79 for H.
The structural units-B-CO-in templates (a1), (a2) and (a3) represent L-amino acid residues. Preferred values for B are: -NR20CH(R71) -and wherein R2A group A5, A8, A22, A25 which is H, wherein R2Is the enantiomer of a38, a42, a47 and a50 of H. Most preferred is
Ala L-alanine
Arg L-arginine
Asn L-asparagine
Cys L-cysteine
Gln L-Glutamine
Gly glycine
His L-histidine
Ile L-isoleucine
Leu L-leucine
Lys L-lysine
Met L-methionine
Phe L-phenylalanine
Pro L-proline
Ser L-serine
Thr L-threonine
Trp L-Tryptophan
Tyr L-tyrosine
Val L-valine
Cit L-citrulline
Orn L-Ornithine
tBuA L-tert-butylalanine
Sar sarcosine
t-BuG L-tert-butylglycine
4AmPhe L-p-aminophenylalanine
3AmPhe L-m-aminophenylalanine
2AmPhe L-O-aminophenylalanine
Phe(mC(NH2) H) L-M-amidinophenylalanine
Phe(pC(NH2) (NH) L-Paramidinophenylalanine
Phe(mNHC(NH2) (NH) L-m-guanidinophenylalanine
Phe(pNHC(NH2) (NH) L-p-guanidinophenylalanine
Phg L-phenylglycine
Cha L-Cyclohexylalanine
C4al L-3-Cyclobutylalanine
C5al L-3-Cyclopentylalanine
Nle L-norleucine
2-Nal L-2-naphthylalanine
1-Nal L-1-naphthylalanine
4Cl-Phe L-4-chlorophenylalanine
3Cl-Phe L-3-chlorophenylalanine
2Cl-Phe L-2-chlorophenylalanine
3，4Cl2-Phe L-3, 4-dichlorophenylalanine
4F-Phe L-4-fluorophenylalanine
3F-Phe L-3-fluorophenylalanine
2F-Phe L-2-fluorophenylalanine
Tic L-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid
Thi L-beta-2-thienylalanine
Tza L-2-Thiazolylalanine
Mso L-methionine sulfoxide
AcLys L-N-acyl lysine
Dpr L-2, 3-diaminopropionic acid
A2Bu L-2，4Diaminobutyric acid
Dbu (S) -2, 3-diaminobutyric acid
Abu gamma-aminobutyric acid (GABA)
Aha epsilon-aminocaproic acid
Aib alpha-aminoisobutyric acid
Y (Bzl) L-O-benzyltyrosine
Bip L-biphenylalanine
S (Bzl) L-O-benzylserine
T (Bzl) L-O-benzylthreonine
hHa L-high cyclohexyl alanine
hCys L-homocysteine
hSer L-homoserine
hArg L-homoarginine
hPhe L-homophenylalanine
Bpa L-4-benzoylphenylalanine
Pip L-pipecolic acid
OctG L-octyl glycine
MePhe L-N-Methylphenylalanine
Menle L-N-Methylnorleucine
MeAla L-N-methylalanine
MeIle L-N-methylisoleucine
MeVal L-N-methylvaline
MeLeu L-N-methylleucine
In addition, the most preferred values of B also include groups of type A8 "in the (L) -configuration:
A8″
wherein R is20Is H or lower alkyl and R64Is an alkyl group; an alkenyl group; - [ (CH)2)u-X]t-CH3(wherein X is-O-; -NR-)20-, or-S-; u-1-3 and t-1-6), aryl; aryl-lower alkyl; or heteroaryl-lower alkyl; in particular wherein R64Is n-hexyl (A8 "-21); n-heptyl (A8 "-22); 4- (phenyl) benzyl (A8 "-23); benzhydryl (A8 "-24); 3-amino-propyl (A8 "-25); 5-amino-pentyl (A8 "-26); methyl (A8' -27); ethyl (A8 "-28); isopropyl (A8 "-29); isobutyl (A8 "-30); n-propyl (A8 "-31); cyclohexyl (A8 "-32); cyclohexylmethyl (A8' -33); n-butyl (A8 "-34); phenyl (A8' -35); benzyl (A8 "-36); (3-indolyl) methyl (A8 "-37); 2- (3-indolyl) ethyl (A8 "-38); (4-phenyl) phenyl (A8 "-39); n-nonyl (A8 "-40); CH (CH)3-OCH2CH2-OCH2- (A8' -41) and CH3-(OCH2CH2)2-OCH2Those of (A8' -42).
The peptide chain Z of the β -hairpin mimetics described herein is generally defined in terms of amino acid residues belonging to one of the following groups:
group C-NR20CH(R72)CO-；“Hydrophobicity: small to medium size "
Group D-NR20CH(R73) CO-; "hydrophobicity: large aromatic or heteroaromatic compounds "
Group E-NR20CH(R74) CO-; "polar-cationic" and "urea-derived"
Group F-NR20CH(R84) CO-; "polar-uncharged or anionic"
Group H-NR20-CH(CO-)-(CH2)4-7-CH(CO-)-NR20-；
-NR20-CH(CO-)-(CH2)pSS(CH2)p-CH(CO-)-NR20-；
-NR20-CH(CO-)-(-(CH2)pNR20CO(CH2)p-CH(CO-)-NR20-; and
-NR20-CH(CO-)-(-(CH2)pNR20CONR20(CH2)p-CH(CO-)-NR20-；
"interchain bond"
Group I-NR86CH2CO-; "polar-cationic or hydrophobic"
Furthermore, the amino acid residues in the chain Z may also have the general formula-A-CO-or the general formula-B-CO-, wherein A and B are as defined above. Finally, Gly can also be an amino acid residue in chain Z, and Pro can also be an amino acid residue in chain Z, except where an interchain bond (H) may be present.
According to the substituent R72Group C includes amino acid residues having hydrophobic side chain groups of small to medium size. Hydrophobic residues refer to amino acid side chains that are uncharged at physiological pH and are repelled by aqueous solutions. In addition, these side chains generally do not contain hydrogen bond donor groups, such as, but not limited to, primaryAnd secondary amides, primary and secondary amines and their corresponding protonated salts, thiols, alcohols, phosphonic acids (salts/esters), phosphoric acids (salts/esters), ureas or thioureas. However, they may contain hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, alkyl-or arylphosphonic acids (salts) and phosphoric acids (salts) or tertiary amines. Genetically encoded small to medium size amino acids include alanine, isoleucine, leucine, methionine and valine.
According to the substituent R73Group D includes amino acid residues having aromatic and heteroaromatic side chain groups. An aromatic amino acid residue refers to a hydrophobic amino acid having a side chain containing at least one ring (aromatic group) with a conjugated pi-electron system. They may additionally contain hydrogen bond donor groups such as, but not limited to, primary and secondary amides, primary and secondary amines and their corresponding protonated salts, thiols, alcohols, phosphonic acids, phosphoric acids, ureas or thioureas, as well as hydrogen bond acceptor groups such as, but not limited to, ethers, thioethers, esters, tertiary amides, alkyl-or arylphosphonic acids and phosphoric acids or tertiary amines. Genetically encoded aromatic amino acids include phenylalanine and tyrosine.
According to the substituent R77By heteroaromatic amino acid residue is meant a hydrophobic amino acid having a side chain containing at least one ring with a conjugated pi-system incorporating at least one heteroatom such as (but not limited to) O, S and N. In addition such residues may contain hydrogen bond donor groups such as, but not limited to, primary and secondary amides, primary and secondary amines and their corresponding protonated salts, thiols, alcohols, phosphonic acids (salts/esters), phosphoric acids (salts/esters), ureas or thioureas, as well as hydrogen bond acceptor groups such as, but not limited to, ethers, thioethers, esters, tertiary amides, alkyl-or arylphosphonic acids (salts/esters) -and phosphoric acids (salts/esters) or tertiary amines. Genetically encoded heteroaromatic amino acids include tryptophan and histidine.
According to the substituent R74Group E includes amino acids having side chains with polar-cationic, amido-or urea-derived residues. Polar-cationic refers to basic side chains that are protonated at physiological pH. Genetic codingThe polar-cationic amino acids of (a) include arginine, lysine and histidine. Citrulline is an example of a urea-derived amino acid residue.
According to the substituent R84Group F includes amino acids containing side chains with polar-uncharged or anionic residues. Polar-uncharged or anionic residues refer to hydrophilic side chains that are uncharged or anionic (carboxylic acids included), respectively, at physiological pH, but are not repelled by aqueous solutions. Such side chains typically contain hydrogen bond donor groups such as, but not limited to, primary and secondary amides, carboxylic acids and esters, primary and secondary amines, thiols, alcohols, phosphonic acids (salts/esters), phosphoric acids (salts/esters), urea or thiourea. These groups are capable of forming a hydrogen bonding network with water molecules. They may additionally contain hydrogen bond acceptor groups such as, but not limited to, ethers, thioethers, esters, tertiary amides, carboxylic acids and carboxylic acids (salts/esters), alkyl-or arylphosphonic acids (salts/esters) and phosphoric acids (salts/esters) or tertiary amines. Genetically encoded polar-uncharged amino acids include asparagine, cysteine, glutamine, serine and threonine, and aspartic acid and glutamic acid.
Group H includes side chains of preferably (L) -amino acids located at opposite positions of the β -strand region capable of forming interchain bonds. The most commonly recognized bond is a disulfide bridge formed by a cysteine and a homo-cysteine, which are located at opposite positions of the β -strand. Various methods are known for forming disulfide bonds, including those described in the following documents: tam et al Synthesis 1979, 955-; stewart et al, Solid Phase Peptide Synthesis, 2d Ed., Pierce chemical Company, III., 1984; ahmed et al J.biol.chem.1975, 250, 8477-8482; and Pennington et al, Peptides, pages 164-. Most advantageously within the scope of the present invention, the disulfide bond may be prepared by an acetamidomethyl (Acm) -protecting group for cysteine. The well-established interchain bond is the attachment of ornithine and lysine to glutamic acid and aspartic acid residues, respectively, at opposite beta-strand positions by amide bond formation. Preferred protecting groups for the side chain amino groups of ornithine and lysine are allyloxycarbonyl (Alloc) and preferred protecting groups for aspartic acid and glutamic acid are allyl esters. Finally, interchain linkages can also be established by linking the amino groups of lysine and ornithine at the opposite β -strand positions to reagents such as N, N-carbonylimidazole to form cyclic ureas.
According to the substituent R86Group I includes glycine having an amino group substituted with a chain containing polar-cationic or hydrophobic residues. Polar-cationic refers to basic side chains that are protonated at physiological pH. Hydrophobic residues refer to amino acid side chains that are repelled by aqueous solutions without electrical charge at physiological pH.
As mentioned previously, the positions of the interchain bonds are: if n is 12, positions P4 and P9; and/or P2 and P11; if n is 14, positions P2 and P13 and/or P4 and P11; if n is 18, the positions P4 and P17 and/or P6 and P15 and/or P8 and P13. Such interchain linkages are known to stabilize the β -hairpin conformation and thus constitute an important structural element in the design of β -hairpin mimetics.
The most preferred amino acid residues in the chain Z are those derived from natural alpha-amino acids. The following is a list of amino acids, or their residues, suitable for the purposes of the present invention, the abbreviations corresponding to the commonly employed convention:
three-letter code and single-letter code
Ala L-alanine A
Arg L-arginine R
Asn L-asparagine N
Asp L-aspartic acid D
Cys L-cysteine C
Glu L-glutamic acid E
Gln L-Glutamine Q
Gly glycine G
His L-histidine H
Ile L-isoleucine I
Leu L-leucine L
Lys L-lysine K
Met L-methionine M
Phe L-phenylalanine F
Pro L-proline P
DPro D-prolineDP
Ser L-serine S
Thr L-threonine T
Trp L-Tryptophan W
Tyr L-tyrosine Y
Val L-valine V
Other α -amino acids, or residues thereof, suitable for the purposes of the present invention include:
cit L-citrulline
Orn L-Ornithine
tBuA L-tert-butylalanine
Sar sarcosine
Pen L-penicillamine
t-BuG L-tert-butylglycine
4AmPhe L-p-aminophenylalanine
3AmPhe L-m-aminophenylalanine
2AmPhe L-o-aminophenylalanine
Phe(mC(NH2) (NH) L-m-amidinophenylalanine
Phe(pC(NH2) (NH) L-p-amidinophenylalanine
Phe(mNHC(NH2) (NH) L-m-guanidinophenylalanine
Phe(pNHC(NH2) (NH) L-p-guanidinophenylalanine
Phg L-phenylglycine
Cha L-Cyclohexylalanine
C4al L-3-Cyclobutylalanine
C5al L-3-Cyclopentylalanine
Nle L-norleucine
2-Nal L-2-naphthylalanine
1-Nal L-1-naphthylalanine
4Cl-Phe L-4-chlorophenylalanine
3Cl-Phe L-3-chlorophenylalanine
2Cl-Phe L-2-chlorophenylalanine
3，4Cl2-Phe L-3, 4-dichlorophenylalanine
4F-Phe L-4-fluorophenylalanine
3F-Phe L-3-fluorophenylalanine
2F-Phe L-2-fluorophenylalanine
Tic 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid
Thi L-beta-2-thienylalanine
Tza L-2-Thiazolylalanine
Mso L-methionine sulfoxide
AcLys N-acetyl lysine
A2Bu 2, 4-diaminobutyric acid
Dbu (S) -2, 3-diaminobutyric acid
Abu gamma-aminobutyric acid (GABA)
Aha epsilon-aminocaproic acid
Aib alpha-aminoisobutyric acid
Y (Bzl) L-O-benzyltyrosine
Bip L- (4-phenyl) phenylalanine
S (Bzl) L-O-benzylserine
T (Bzl) L-O-benzylthreonine
hHa L-high cyclohexyl alanine
hCys L-homocysteine
hSer L-homoserine
hArg L-homoarginine
hPhe L-homophenylalanine
Bpa L-4-benzoylphenylalanine
4-AmPyrr1 (2S, 4S) -4-amino-pyrrolidine-L-carboxylic acid
4-AmPyrr2 (2S, 4R) -4-amino-pyrrolidine-L-carboxylic acid
4-Phepyrr1 (2S, 5R) -4-phenyl-pyrrolidine-L-carboxylic acid
4-Phepyrr2 (2S, 5S) -4-phenyl-pyrrolidine-L-carboxylic acid
5-Phepyrr1 (2S, 5R) -5-phenyl-pyrrolidine-L-carboxylic acid
5-Phepyrr2 (2S, 5S) -5-phenyl-pyrrolidine-L-carboxylic acid
Pro (4-OH)1 (4S) -L-hydroxyproline
Pro (4-OH)2 (4R) -L-hydroxyproline
Pip L-pipecolic acid (pipecolic acid)
DPip D-pipecolic acid (pipecolic acid)
OctG L-octyl glycine
NGly N-methylglycine
MePhe L-N-Methylphenylalanine
Menle L-N-Methylnorleucine
MeAla L-N-methylalanine
MeIle L-N-methylisoleucine
MeVal L-N-methylvaline
MeLeu L-N-methylleucine
DimK L- (N ', N' -dimethyl) -lysine
Lpzp L-piperazinic acid
Dpzp D-piperazinic acid
Isorn L- (N ', N' -diisobutyl) -ornithine
PipAla L-2- (4' -piperidinyl) -alanine
Pirra Ala L-2- (3' -pyrrolidinyl) -alanine
Ampc 4-amino-piperidine-4-carboxylic acid
NMeR L-N-methyl arginine
NMeK L-N-methyllysine
NMePhe L-N-Methylphenylalanine
IPegK L-2-amino-6- {2- [2- (2-methoxyethoxy) ethoxy ]
Acetylamino } -hexanoic acid
SPegK L-2-amino-6- [2- (2-methoxy-ethoxy) -acetamido-
Yl-hexanoic acid
Dab L-1, 4-diamino-butyric acid
IPegdab L-2-amino-4- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethoxy-
Yl acetylamino } -butyric acid
Spegdab L-2-amino-4- [2- (2-methoxy-ethoxy) -acetamido-
Yl ] butanoic acid
4-PyrAla L-2- (4' -pyridyl) -alanine
OrnPyr L-2-amino-5- [ (2' -carbonylpyrazine) ] aminopentanoic acid
BnG N-benzylglycine
(4-OH) BnG N-4-hydroxy-benzylglycine
IaG N-isoamyl glycine
IbG N-isobutyl glycine
(EA) G N- (2-aminoethyl) glycine
(PrA) G N- (3-amino-n-propyl) glycine
(BA) G N- (4-amino-n-butyl) glycine
(PeA) G N- (5-amino-n-pentyl) glycine
(PEG3-NH2)G N-[(CH2)3O-(CH2-CH2O)2-(CH2)3-NH2]Glycine
(Pyrr) G N- {2- [2 '- (1' -methyl-pyrrolidinyl) ] -ethyl } -glycine
(Dimp) G N- [2- (N ', N' -dimethylamino) -propyl ] -glycine
(Im) G N- [3- (1' -imidazolyl) -propyl ] -glycine
(Pip) G N- {3- [1 '- (4' -methylpiperazinyl) ] -propyl } -glycine
(Dime) G N- [2- (N ', N' -dimethylamino) -ethyl ] -glycine
Particularly preferred residues of group C are:
ala L-alanine
Ile L-isoleucine
Leu L-leucine
Met L-methionine
Val L-valine
tBuA L-tert-butylalanine
t-BuG L-tert-butylglycine
Cha L-Cyclohexylalanine
C4al L-3-Cyclobutylalanine
C5al L-3-Cyclopentylalanine
Nle L-norleucine
hHa L-high cyclohexyl alanine
OctG L-octyl glycine
MePhe L-N-Methylphenylalanine
Menle L-N-Methylnorleucine
MeAla L-N-methylalanine
MeIle L-N-methylisoleucine
MeVal L-N-methylvaline
MeLeu L-N-methylleucine
Particularly preferred residues of group D are:
his L-histidine
Phe L-phenylalanine
Trp L-Tryptophan
Tyr L-tyrosine
Phg L-phenylglycine
2-Nal L-2-naphthylalanine
1-Nal L-1-naphthylalanine
4Cl-Phe L-4-chlorophenylalanine
3Cl-Phe L-3-chlorophenylalanine
2Cl-Phe L-2-chlorophenylalanine
3，4Cl2-Phe L-3, 4-dichlorophenylalanine
4F-Phe L-4-fluorophenylalanine
3F-Phe L-3-fluorophenylalanine
2F-Phe L-2-fluorophenylalanine
Thi L-beta-2-thienylalanine
Tza L-2-Thiazolylalanine
Y (Bzl) L-O-benzyltyrosine
Bip L-biphenylalanine
S (Bzl) L-O-benzylserine
T (Bzl) L-O-benzylthreonine
hPhe L-homophenylalanine
Bpa L-4-benzoylphenylalanine
Pirra Ala L-2- (3' -pyrrolidinyl) -alanine
NMePhe L-N-Methylphenylalanine
4-PyrAla L-2- (4' -pyridyl) -alanine
Particularly preferred residues of group E are:
arg L-arginine
Lys L-lysine
Orn L-Ornithine
Dpr L-2, 3-diaminopropionic acid
Dbu (S) -2, 3-diaminobutyric acid
Phe(pNH2) L-p-aminophenylalanine
Phe(mNH2) L-m-aminophenylalanine
Phe(oNH2) L-o-aminophenylalanine
hArg L-homoarginine
Phe(mC(NH2) (NH) L-m-amidinophenylalanine
Phe(pC(NH2) (NH) L-p-amidinophenylalanine
Phe(mNHC(NH2) (NH) L-m-guanidinophenylalanine
Phe(pNHC(NH2) (NH) L-p-guanidinophenylalanine
DimK L- (N ', N' -dimethyl) -lysine
Isorn L- (N ', N' -diisobutyl) -ornithine
NMeR L-N-methyl arginine
NMeK L-N-methyllysine
IPegK L-2-amino-6- {2- [2- (2-methoxyethoxy) ethoxy ]
Acetylamino } -hexanoic acid
Spegk L-2-amino-6- [2- (2-methoxy-ethoxy) -acetylamino
Yl-hexanoic acid
Dab L-1, 4-diamino-butyric acid
IPegdab L-2-amino-4- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethoxy-
Yl acetylamino } -butyric acid
Spegdab L-2-amino-4- [2- (2-methoxy-ethoxy) -acetamido-
Yl ] butanoic acid
OrnPyr L-2-amino-5- [ (2' -carbonylpyrazine) ] aminopentanoic acid
PipAla L-2- (4' -piperidinyl) -alanine
Particularly preferred residues of group F are:
asn L-asparagine
Asp L-aspartic acid
Cys L-cysteine
Gln L-Glutamine
Glu L-glutamic acid
Ser L-serine
Thr L-threonine
Cit L-citrulline
Pen L-penicillamine
AcLys L-N epsilon-acetyl lysine
hCys L-homocysteine
hSer L-homoserine
Particularly preferred residues of group I are
(EA) G N- (2-aminoethyl) glycine
(PrA) G N- (3-amino-n-propyl) glycine
(BA) G N- (4-amino-n-butyl) glycine
(PeA) G N- (5-amino-n-pentyl) glycine
(EGU) G N- (2-guanidinoethyl) glycine
(PrGU) G N- (3-guanidino-n-propyl) glycine
(BGU) G N- (4-guanidino-n-butyl) glycine
(PeGU) G N- (5-guanidino-n-pentyl) glycine
(PEG3-NH2)G N-[(CH2)3O-(CH2-CH2O)2-(CH2)3-NH2]Glycine
(Pyrr) G N- {2- [2 '- (1' -methyl-pyrrolidinyl) ] -ethyl } -glycine
(Dimp) G N- [2- (N ', N' -dimethylamino) -propyl ] -glycine
(Im) G N- [3- (1' -imidazolyl) -propyl ] -glycine
(Pip) G N- {3- [1 '- (4' -methylpiperazinyl) ] -propyl } -glycine
(Dime) G N- [2- (N ', N' -dimethylamino) -ethyl ] -glycine
Typically, the peptide chain Z within the β -hairpin mimetics of the invention comprises 12, 14 or 18 amino acid residues. The individual amino acid residues at positions P1 to P12 or respectively to P14 or P18 in the chain Z are explicitly defined as follows: p1 represents the first amino acid in chain Z which is coupled N-terminally to the C-terminus of template (B) - (P) or of a group-B-CO-in template (a1) or of a group-A-CO-in template (a2) or of a group-B-CO-forming the C-terminus of template (a 3); p12 or, respectively, P14 or P18 represents the last amino acid in the chain Z which is coupled C-terminally to the N-terminus of template (B) - (P) or of the group-A-CO-in template (a1) or of the group-B-CO-in template (a2) or of the group-B-CO-forming the N-terminus of template (a 3). As shown below, each of the positions P1 to P12 or, respectively, to P14 or P18 preferably contains an amino acid residue belonging to one of the above types C, D, E, F, I, H or having the formula-A-CO-or having the formula-B-CO-or being Gly, NMeGly, Pro or Pip:
if n is 12, the α -amino acid residues in positions 1 to 12 of the chain Z are preferably:
-P1: belonging to type C, or type D, or type F, or the residue is Pro or Pip;
-P2: belonging to type E, or type F, or the residue is Gly, NMeGly, Pro or Pip;
-P3: belonging to type E, or type F;
-P4: belonging to type C, or type D, or type F, or the residue is Gly or NMeGly;
-P5: belonging to type E, or type D, or type F, or the residue is Gly, NMeGly, Pro or Pip;
-P6: belonging to type E, or type F, or having the general formula-B-CO-, or the residue is Gly or NMeGly;
-P7: belonging to type E, or type F;
-P8: is of type D, or type C, or the residue is Pro or Pip;
-P9: belonging to type C, or type D, or type F, or the residue is Gly or NMeGly;
-P10: is of type D, or type C, or the residue is Pro or Pip;
-P11: belonging to type E, or type F, or the residue is Gly or NMeGly;
-P12: belonging to type E or type F, or the residue is Pro or Pip; or
-P4 forms together with P9 a group of type H;
d-isomers at P4, P6, P9 are also possible.
If n is 12, the α -amino acid residues at positions 1 to 12 are most preferably:
-P1：Tyr；
-P2：Arg，Gly；
-P3：Cit；
-P4：Val，Phe，Gly，Ile，Thr，Gln，Cys；
-PS：Arg；
-P6：Arg，DArg；
-P7：Arg；
-P8：Trp，2-Nal；
-P9：Val，Phe，Gly，Ile，Thr，Gln，Cys；
-P10：Tyr；
-P11: cit, Gly; and
-P12: lys; or
-disulfide bond formation at P4 with Cys at P9.
If n is 14, the α -amino acid residues in positions 1 to 14 of the chain Z are preferably:
-P1: belonging to type C, or type D, or type E, or type F, or the residue is Gly or NMeGly or Pro or Pip;
-P2: belonging to type E, or type D, or type F;
-P3: belonging to type E, or type F, or type D, or type C, or the residue is Pro or Pip;
-P4: belonging to type D, or type C, or type F;
-P5: belonging to type E, or type F, or type I;
-P6: belonging to type C, or type D, or type F, or the residue is Gly, NMeGly, Pro or Pip;
-P7: belonging to type C, or type D, or having the general formula-A-CO-, or the residue is Gly, NMeGly, Pro or Pip;
-P8: belonging to type E, or type F, or type D, or type I, or the residue is Pro or Pip;
-P9: belonging to type F, or type E, or type D, or type I, or the residue is Pro or Pip;
-P10: belonging to type F, or type D, or type C;
-P11: belonging to type D, or type C, or type F, or type E;
-P12: belonging to type C, or type D, or type F;
-P13: belonging to type F, or type E, or type D, or type C, or type I, or the residue is Gly or NMeGly;
-P14: belonging to type F, or type E, or type C; or
-P2 and P13 and/or P4 and P11 together form a group of type H;
d-isomers at position P4, P7, P8 or P11 are possible;
provided that
-the amino acid residue at position P1 is Gly or NMeGly or Pip; and/or
-the amino acid residue at position P2 belongs to type F; and/or
-the amino acid residue at position P3 belongs to type F, or it is Pro or Pip; and/or
-the amino acid residue at position P4 belongs to type F; and/or
-the amino acid residue at position P5 belongs to type F, or type I; and/or
-the amino acid residue at position P6 belongs to type C, or type D, or it is NMeGly or Pip; and/or
-the amino acid residue at position P7 belongs to type C, or type D, or it is NMeGly, Pro or Pip;
and/or
The amino acid residue at position P8 is of type D, or of type I, or it is Pro or Pip and/or
-the amino acid residue at position P9 belongs to type F, or type I, or it is Pip; and/or
-the amino acid residue at position P10 belongs to type F; and/or
-the amino acid residue at position P11 belongs to type C; and/or
-the amino acid residue at position P12 belongs to type C, or type F; and/or
-the amino acid residue at position P13 belongs to type F, or it is Gly or NMeGly; and/or
-P4 and P11 together form a group of type H; and/or
-the amino acid residue at position P4 is the D-isomer; and/or
-the amino acid residue at position P11 is the D-isomer.
If n is 14, the α -amino acid residues at positions 1 to 14 are most preferably:
-P1：Tyr，Gln，Arg，His，Ile，Trp，Thr，Glu，Ser，Val，Met，Phe，Gly，Asp，Leu，Pip；
-P2：Arg，His，Lys，4-PyrAla；
-P3：Cit；Arg，His，Ile，Tyr，Trp，Pro，Glu，Asn，Asp，Lys，Ala，Leu，Val，4F-Phe，Met，Ser，Thr，Gln，Tyr；
-P4：Val，Phe，Tyr，t-BuG，Cys，Ser，Dab，Glu；
-P5：Arg，Dab，Ser，(EA)G；
-P6：Pro，Gly，Phe，Val，Cit，Ala；
-P7：DPro，Pro，Gly，Val；
-P8：Arg，Tyr，Trp，Thr，4F-Phe，Dab，4-PyrAla，Isorn，(Im)G，Cit，His，IpegDab，DPro；
-P9：Arg，(Pip)G，(EA)G，Orn，Pro；
-P10：2-Nal，Trp，Tyr；
-P11：Phe，Tyr，Val，t-BuG，Cys，Asn，Glu，Dab，Arg；
-P12：Tyr，Cit；
-P13：Cit，Gln，Arg，His，Tyr，Asn，Asp，Lys，Ala，Ser，Leu，Met，NMeGly，Thr，Cys；
-P14：Lys，Glu，Gln，Asn，Asp，Ala，Ser，NMeK；
provided that
-the amino acid residue at position P1 is Pip or Gly; and/or
-the amino acid residue at position P3 is Glu, Asn, Asp, Thr, or gin; and/or
-the amino acid residue at position P4 is Cys, Ser or Glu; and/or
-the amino acid residue at position P5 is Ser or (EA) G; and/or
-the amino acid residue at position P6 is Phe, Val or Ala; and/or
-the amino acid residue at position P7 is Val, Pro, orDPro; and/or
-the amino acid residue at position P8 is Tyr, Trp, 4F-Phe, 4-PyrAla, (Im) G, His orDPro; and/or
-the amino acid residue at position P9 is (EA) G; and/or
-the amino acid residue at position P10 is Val or t-BuG; and/or
-the amino acid residue at position P12 is Tyr or Cit; and/or
-the amino acid residue at position P13 is Glu, gin, Asp, Asn, Ser, Thr, Cys or NMeGly; and/or
-Cys at P4 and P11 forms a disulfide bond; and/or
-Glu at P4 forming a lactam bond with Dab at P11; and/or
Dab at P4 forming a lactam bond with Glu at P11.
If n is 18, the amino acid residues in positions 1-18 are preferably:
-P1: belonging to type D, or type E;
-P2: belonging to type E, or type F;
-P3: is of type C, or type D;
-P4: belonging to type E, or type F;
-P5: belonging to type D, or type E;
-P6: belonging to type E, or type F;
-P7: belonging to type E, or type F;
-P8: belonging to type E, or type F, or the residue is Gly or NMeGly;
-P9: belongs to type D;
-P10: is of type E, or has the general formula-A1-A69-CO-, or the residue is Pro or Pip;
-P11: belonging to type E, or having the general formula-B-CO-, or the residue is Gly, NMeGly, Pro or Pip;
-P12: belongs to type D;
-P13: belonging to type F, or type E, or the residue is Gly or NMeGly;
-P14: is of type C, or type D;
-P15: belonging to type E, or type F;
-P16: belonging to type E or type F;
-P17: belonging to type E, or type F;
-P18: belonging to type C or type D or type E or type F; or
-P4 and P17 and/or P6 and P15 and/or P8 and P13 together form a group of type H;
d-isomers at positions P10, P11 and P12 are also possible.
If n is 18, the α -amino acid residues in positions 1 to 18 are most preferably:
-P1：Arg；
-P2：Arg；
-P3：2-Nal，Trp，Tyr；
-P4：Cys；
-P5：Tyr；
-P6：Cit，Gln.Arg；
-P7：Lys；
-P8：Cys，Gly；
-P9：Tyr；
-P10：Lys，DLys，DPro；
-P11：Gly，Pro，DPro；
--P12：Tyr；
-P13：Cys，Gly；
-P14：Tyr；
-P15：Arg；
-P16：Cit，Thr，Lys；
-P17: cys; and
-P18: arg; or
-disulfide bond formation between P4 and P17 and/or between P8 and Cys at P13.
Particularly preferred β -peptide mimetics of the invention include those described in examples 21, 22, 38, 45, 51, 5253, 55, 56, 60, 61, 68, 75, 84, 85, 87, 101, 102, 105, 110, 120, 132, 147, 151, 152 and 160.
The method of the invention can advantageously be carried out in a parallel array synthesis, resulting in a library of template-fixed β -hairpin peptidomimetics of the general formula I above. This parallel synthesis allows arrays (arrays) of many (usually 24 to 192, typically 96) compounds of formula I to be obtained in high yields and with a defined purity, minimizing the formation of dimeric and multimeric by-products. The proper selection of functionalized solid support (i.e., solid support plus linker molecule), template, and cyclization sites therefore plays an important role.
The functionalized solid support may conveniently be selected from polystyrene crosslinked with (preferably 1-5%) divinylbenzene; coated with polyethylene glycol spacer (Tentagel)R) The polystyrene of (4); and polyacrylamide resin derivatization (see also Obrecht, D.; Villalgord, J. -M, "Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compounds", Tetrahedron organic chemistry Series, Vol.17, Pergamon, Elsevier Science, 1998).
The solid support is functionalized with a linker (linker), a bifunctional spacer molecule containing a binding group at one end for attachment to the solid support and a selectively cleavable functional group at the other end for subsequent chemical transformation and cleavage processes. For the purposes of the present invention, two types of linker are used:
type 1 linkers are designed to release the amide group under acidic conditions (Rink H, tetrahedron Lett.1987, 28, 3783-. This type of linker forms an amide of the carboxyl group of the amino acid; examples of resins functionalized by this linker structure include 4- [ ((((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] PS resin, 4- [ (((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] -4-methylbenzhydrylamine PS resin (Rink amide MBHA PS resin), and 4- [ ((((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] benzhydrylamine PS-resin (Rink amide BHA PS resin). Preferably, the support is derived from polystyrene cross-linked with (most preferably 1-5%) divinylbenzene and functionalized with a 4- (((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) linker.
Type 2 linkers are designed to eventually release the carboxyl group under acidic conditions. This type of linker forms acid labile esters with the carboxyl group of amino acids, typically acid labile benzyl, benzhydryl and trityl esters; examples of such linker structures include 2-methoxy-4-hydroxymethylphenoxy (Sasrin)RLinker), 4- (2, 4-dimethoxyphenyl-hydroxymethyl) -phenoxy (Rink linker), 4- (4-hydroxymethyl-3-methoxyphenoxy) butanoic acid (HMPB linker), trityl and 2-chlorotrityl. Preferably, the support is derived from polystyrene cross-linked with (most preferably 1-5%) divinylbenzene and functionalized with a 2-chlorotrityl linker.
When performed as a parallel array synthesis, the process of the invention may advantageously be performed as described below, but it will be apparent to the skilled person how these procedures may be modified when it is desired to synthesize a single compound of formula I above.
In a number of reaction vessels equal to the total number of compounds to be synthesized by the parallel method (generally 24 to 192, typically 96) 25-1000mg, preferably 100mg, of a suitable functionalized solid support, preferably 1-3% cross-linked polystyrene or Tentagel resin, are loaded.
The solvent used is capable of swelling the resin and includes, but is not limited to, Dichloromethane (DCM), Dimethylformamide (DMF), N-methylpyrrolidone (NMP), dioxane, toluene, Tetrahydrofuran (THF), ethanol (EtOH), Trifluoroethanol (TFE), isopropanol, and the like. Solvent mixtures containing at least one component as polar solvent (e.g. 20% TFE/DCM, 35% THF/NMP) are beneficial for ensuring high reactivity and solvation of the peptide chain bound to the resin (Fields, g.b., Fields, c.g., j.am.chem.soc.1991, 113, 4202-4207).
With the development of various linkers for releasing the C-terminal carboxylic acid group under mildly acidic conditions without affecting the acid labile group protecting the functional group in the side chain, significant progress has been made in the synthesis of protected peptide fragments. 2-methoxy-4-hydroxybenzyl alcohol-derived linker (Sasrin)RLinker, Mergler et al Tetrahedron Lett.1988, 29, 4005-4008) was cleavable with dilute trifluoroacetic acid (0.5-1% TFA in DCM) and was stable under Fmoc deprotection conditions during peptide synthesis, and other protecting groups based on Boc/tBu were compatible with this protection scheme. Other linkers suitable for the method of the invention include the superacid labile 4- (2, 4-dimethoxyphenyl-hydroxymethyl) -phenoxy linker (Rink linker, Rink, h. tetrahedron lett.1987, 28, 3787-3790) -wherein removal of the peptide requires 10% concentration of acetic acid in DCM or 0.2% concentration of trifluoroacetic acid in DCM; 4- (4-hydroxymethyl-3-methoxyphenoxy) butanoic acid-derived linker (HMPB-linker, Fl rsheimer)&Riniker, Peptides 1991, 1990, 131) -which can also be cleaved with 1% TFA/DCM to give peptide fragments containing all acid labile side chain protecting groups; and, in addition, a 2-chlorotriphenylmethyl chloride linker (Barlos et al, Tetrahedron Lett.1989, 30, 3943-.
Suitable protecting groups for amino acids or their residues are, for example,
for amino groups (e.g. also in the side chain of lysine)
Cbz benzyloxycarbonyl
Boc tert-butoxycarbonyl
Fmoc 9-fluorenylmethoxycarbonyl
Alloc allyloxycarbonyl radical
Teoc trimethylsilyl ethoxycarbonyl
Tc Trichloroethoxycarbonyl
Nps o-nitrophenylsulfonyl;
trt triphenylmethyl or trityl
For carboxyl groups (for example also present in the side chains of aspartic acid and glutamic acid), by conversion into esters with an alcohol component
tBu tert-butyl
Bn benzyl group
Me methyl group
Ph phenyl
Pac phenacyl
Allyl radical
Tse Trimethylsilylethyl group
Tce trichloroethyl;
for guanidino (e.g. present in the side chain of arginine)
Pmc 2, 2, 5, 7, 8-pentamethyl chroman-6-sulfonyl
Ts tosyl (i.e. p-tosyl)
Cbz benzyloxycarbonyl
Pbf methyl dihydrobenzofuran-5-sulfonyl
For hydroxyl groups (e.g. present in the side chains of threonine and serine)
tBu tert-butyl
Bn benzyl group
Trt trityl radical
For thiol groups (e.g. present in the side chain of cysteine)
Acm acylamidomethyl group
tBu tert-butyl
Bn benzyl group
Trt trityl radical
Mtr 4-methoxytrityl.
The 9-fluorenylmethoxycarbonyl- (Fmoc) -protected amino acid derivative is preferably used as building block for the construction of template-fixed beta-hairpin loop mimetics of the general formula I. For deprotection, i.e. cleavage of the Fmoc group, 20% piperidine in DMF or 2% DBU/2% piperidine in DMF may be used.
N-substituted glycine derivatives (type I) useful as building blocks for certain compounds of the general formula I can be derived from 9-fluorenylmethoxycarbonyl- (Fmoc) -protected amino acid derivatives or can be derived in two steps from glycine precursors containing a leaving group, such as bromo-, chloro-or iodoacetic acid, and the appropriate primary amine building block NH2-R86And (4) forming. The first synthetic step is that an acetylating agent containing a leaving group, such as bromoacetic acid, is attached to the resin bound intermediate through the formation of an amide bond. The second reaction step, nucleophilic displacement, is accomplished by using primary amine building blocks, wherein the residue is, if necessary, suitably protected with a group as described above for the side chain of the amino acid.
For the introduction of N-substituted glycine derivatives as building blocks into template-immobilized β -hairpin loop mimetics, the general synthetic methods for assembling hairpin mimetics are used as described herein.
The amount of reactants, i.e. amino acid derivatives, is usually 1-20 equivalents (based on milliequivalents per gram (meq/g) of functionalized solid support initially weighed into the reaction tube (typically 0.1-2.85 milliequivalents per g for polystyrene resins)). Additional equivalents of reactants can be used to drive the reaction to completion in a reasonable time, if desired. The reaction tubes, together with a holder (hold block) and a manifold (maniffold), were reinserted into a reservoir block (reservoirblock) and the device was then fastened together. Gas is flowed through the manifold to provide a controlled environment, such as nitrogen, argon, air, and the like. The gas stream may also be heated or cooled prior to flowing through the manifold. Heating or cooling of the reaction well is achieved by heating the reaction mass or external cooling with isopropanol/dry ice or the like to cause the desired synthesis reaction. Stirring is achieved by shaking or magnetic stirring (within the reaction tube). Preferred workstations, however not limited to, are the Labsource's Combi-chem workstation and the MultiSynTech's-Syro synthesizer.
Amide bond formation requires activation of the alpha-carboxyl group for this acylation step. When this activation is carried out using a conventionally used carbodiimide-based compound such as dicyclohexylcarbodiimide (DCC, Sheehan & Hess, J.Am.chem.Soc.1955, 77, 1067-containing 1068) or diisopropylcarbodiimide (DIC, Saratakis et al biochem.Biophys.Res.Commun.1976, 73, 336-containing 342), the obtained dicyclohexylurea and diisopropylurea are insoluble or soluble, respectively, in a conventionally used solvent. In a variant of the carbodiimide process, 1-hydroxybenzotriazole (HOBt, K _ nig & Geiger, chem. Ber 1970, 103, 788-. HOBt prevents dehydration, inhibits racemization of activated amino acids, and acts as a catalyst to improve slow coupling reactions. Certain phosphonium reagents have been used as direct coupling reagents, such as benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP, Castro et al, Tetrahedron Lett.1975, 14, 1219-; these phosphonium reagents are also suitable for forming HOBt esters in situ with protected amino acid derivatives. More recently, diphenoxyphosphoryl azide (DPPA) or O- (7-aza-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate (TATU) or O- (7-aza-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU)/7-aza-1-hydroxybenzotriazole (HOAt, Carpino et al, Tetrahedron Lett.1994, 35, 2279-.
Since a near quantitative coupling reaction is necessary, experimental evidence of reaction completion is desired. The ninhydrin test (Kaiser et al, anal. biochemistry 1970, 34, 595), in which a positive colorimetric reaction on an aliquot of resin-bound peptide qualitatively indicates the presence of a primary amine, can be readily and rapidly performed after each coupling step. Fmoc chemistry allows for spectrophotometric detection of Fmoc chromophores when they are released with a base (Meienhofer et al, int.j. peptide Protein res.1979, 13, 35-42).
The resin bound intermediates in each reaction tube were washed to remove remaining excess reagents, solvents and byproducts by repeated exposure to pure solvent according to one of two following methods:
1) adding a solvent (preferably 5ml) to the reaction well, immersing the reaction tube with the holder and manifold therein and stirring for 5-300 minutes, preferably 15 minutes, and then draining the liquid by gravity and then applying gas pressure through the manifold inlet (while closing the outlet) to expel the solvent;
2) the manifold is removed from the jig and an equal volume of solvent (preferably 5ml) is dispensed through the top of each reaction tube and drained by gravity through a filter into a receiving container such as a test tube or vial.
The two above washing procedures are repeated no more than about 50 times (preferably about 10 times), and the efficiency of removing reagents, solvents and by-products is monitored by methods such as TLC, GC or inspection of washings.
The above procedure of reacting the resin bound compound with a reagent and then removing excess reagent, byproducts and solvent in the reaction well is repeated to achieve each successive conversion until the final resin bound fully protected linear peptide is obtained.
Before this fully protected linear peptide is separated from the solid support, one or several protected functional groups present in the molecule can be optionally deprotected, if desired, and the reactive groups thus released can be appropriately replaced. For this purpose, the functional group must be initially protected by a protecting group which can be selectively removed without affecting the presence of the remaining protecting group. Alloc (allyloxycarbonyl) is an example of such an amino protecting group which can be used, for example, in CH2Cl2Pd ° and phenylsilane are selectively removed without affecting the remaining protecting groups such as Fmoc present in the molecule. The released reactive groups may then be substituted as appropriate to introduce the desired substitutionThe reagent of the base is processed. Thus, for example, an amino group can be acylated with an acylating agent corresponding to the acyl substituent to be introduced. For the formation of a pegylated amino acid, such as IPegK or SPegK, it is preferred to let 5 equivalents of HATU (N- [ (dimethylamino) -1H-1, 2, 3-triazolo [4, 5-b ]]Pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide) solution in dry DMF and 10 equivalents of DIPEA (diisopropylethylamine) solution in dry DMF were separately mixed with 5 equivalents of 2- [2- (2-methoxyethoxy) ethoxy ] ethoxy]Acetic acid (1Peg) or 2- (2-methoxyethoxy) acetic acid (sPeg) acts on the amino group of the side chain of the appropriate amino acid released for 3 hours. After filtration and washing of the resin the procedure was repeated for another 3h with a new reagent solution.
Before this fully protected linear peptide is detached from the solid support, interchain bonds can also be formed, if desired, between the side chains of suitable amino acid residues located at opposite positions in the β -chain region.
Interchain bonds and their formation have been discussed above by the explanations given in relation to the group of type H, which may be, for example, disulfide bonds formed by cysteine and homocysteine residues in opposite positions of the β -chain; or a lactam bond formed by the amide bond formation of an ornithine residue and a lysine residue linked to a glutamic acid residue and an aspartic acid residue, respectively, at opposite beta-chain positions or a2, 4-diaminobutyric acid residue linked to a glutamic acid residue at opposite beta-chain positions. The formation of such interchain bonds can be carried out by methods well known in the art.
For disulfide bond formation, preferably, 10 equivalents of iodine solution in DMF or in CH2Cl2The reaction in the MeOH/MeOH mixture was carried out for 1.5 h (this was repeated with fresh iodine solution for another 3h after filtration of the iodine solution), or else after having been carried out with 5% NaHCO3A mixture of DMSO and acetic acid buffered to pH5-6 was allowed to act for 4 hours, or by stirring in water adjusted to pH8 with ammonium hydroxide solution for 24 hours, or in a solution of NMP and tri-n-butylphosphine (preferably 50 equivalents).
For the formation of the lactam bond, preferably a solution of 2 equivalents of HATU (N- [ (dimethylamino) -1H-1, 2, 3-triazolo [4, 5-b ] pyridin-1-ylmethylene ] -N-methylmethanaminium hexafluorophosphate N-oxide) in dry DMF and a solution of 4 equivalents of DIPEA (diisopropylethylamine) in dry DMF are applied for 16 hours.
The detachment of the fully protected linear peptide from the solid support is achieved by dipping the reaction tube together with a clamp and manifold into a reaction well containing a solution of the cleaving reagent (preferably 3 to 5 ml). Gas flow, temperature control, agitation and reaction monitoring were performed as described above and the disengagement reaction was carried out as needed. The reaction tube is removed from the receptacle block with the clamp and manifold and lifted above the liquid level but below the upper lip (upper lip) of the reaction well, and then air pressure is applied through the manifold inlet (while closing the outlet) to effectively drain the final product solution into the receptacle well. The resin remaining in the reaction tube is then washed 2-5 times with 3-5ml of a suitable solvent as described above to extract (wash out) as much of the liberated product as possible. The product solutions obtained were combined, taking care to avoid cross-mixing. Each solution/extract is then treated as necessary to isolate the final compound. Typical treatments include, but are not limited to, evaporation, concentration, liquid/liquid extraction, acidification, basification, neutralization or other reactions in solution.
The solution containing the fully protected linear peptide derivative which has been cleaved from the solid support and neutralized with a base is subjected to evaporation. The cyclization is then carried out in solution by using a solvent such as DCM, DMF, dioxane, THF or the like. Various coupling reagents as mentioned above can be used for this cyclization. The duration of the cyclization is about 6 to 48 hours, preferably about 16 hours. The progress of the reaction is followed, for example, by RP-HPLC (reverse phase high performance liquid chromatography). The solvent is then removed by evaporation, the fully protected cyclic peptide derivative is dissolved in a water-immiscible solvent such as DCM, and the resulting solution is extracted with water or a mixture of water-miscible solvents in order to remove any excess coupling reagent.
Alternatively, detachment and complete deprotection of the fully protected peptide from the solid support can be achieved manually in a glass container.
Finally, the fully protected peptide derivative was treated with 95% TFA, 2.5% H20, 2.5% TIS or other scavenger combination treatment to effect cleavage of the protecting group. The cleavage reaction time is generally 30 minutes to 12 hours, preferably about 2.5 hours. The volatiles were evaporated to dryness, then the crude peptide was dissolved in 20% AcOH aqueous solution and extracted with diisopropyl ether or other solvent suitable for the purpose. The aqueous layer was collected and evaporated to dryness and the fully deprotected cyclic peptide derivative of general formula I was obtained as the final product. Depending on its purity, these peptide derivatives can be used directly in biological tests, or require further purification, for example by preparative HPLC.
If desired, the fully deprotected product of the general formula I obtained can then be converted into a pharmaceutically acceptable salt or the pharmaceutically acceptable or unacceptable salt obtained can be converted into the corresponding free compound of the general formula I or into a different pharmaceutically acceptable salt, as described earlier. Any of these operations can be performed by methods known in the art.
The template starting materials of the general formula II used in the process of the present invention, the starting materials used therefor, and the methods for the preparation of these starting and starting materials have been described in the international application PCT/EP02/01711 published as WO 02/070547A 1 by the same applicant.
General formula H2NR86The starting materials of (a) are known or can be prepared by methods well known in the art.
The beta-hairpin peptidomimetics of the invention can be used in a wide variety of applications in order to prevent HIV infection in healthy individuals and slow or stop the progression of the virus in infected patients, or for the treatment of immunosuppression when cancer is mediated by CXCR4 receptor activity or by CXCR4 receptor activity, or when immunological diseases are caused or mediated by CXCR4 receptor activity, or they can be used during the reinjection collection of peripheral blood stem cells.
The β -hairpin peptidomimetic may be administered as such or may be administered in a suitable formulation with carriers, diluents or excipients well known in the art.
When used to treat or prevent HIV infection or cancer such as breast cancer, brain cancer, prostate cancer, lung cancer, kidney cancer, neuroblastoma, non-hodgkin's lymphoma, ovarian cancer, multiple myeloma, chronic lymphocytic (lymphocytic) leukemia, pancreatic cancer, melanoma, angiogenesis, and hematopoietic tissues; or inflammatory disorders such as asthma, allergic rhinitis, hypersensitivity lung disease, hypersensitivity pneumonitis, eosinophilic pneumonia, delayed hypersensitivity, Interstitial Lung Disease (ILD), idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing sponylitis, systemic sclerosis, Sjogren's syndrome, systemic hypersensitivity or hypersensitivity reactions, drug allergies, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes, glomerulonephritis, autoimmune throiditis, transplant rejection including allograft rejection or graft-versus-host disease, inflammatory bowel disease, inflammatory skin diseases; or for the treatment of immunosuppression, including immunosuppression induced by chemotherapy, radiotherapy or graft/transplantation rejection, the beta-hairpin peptidomimetics can be administered alone, as a mixture of several beta-hairpin peptidomimetics, in combination with other anti-HIV agents, or antimicrobial or anti-cancer or anti-inflammatory agents, or in combination with other pharmaceutically active agents. The β -hairpin peptidomimetics can be administered as such or in the form of a pharmaceutical composition.
Pharmaceutical compositions comprising the β -hairpin peptidomimetics of the invention may be manufactured by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping (entrap) or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active β -hairpin peptidomimetics into preparations which can be used pharmaceutically. The appropriate formulation depends on the method of administration chosen.
For topical administration, the β -hairpin peptidomimetics of the invention can be formulated into solutions, gels, ointments, creams, suspensions, and the like, as are well known in the art.
Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.
For injections, the β -hairpin peptidomimetics of the invention can be formulated in a suitable solution, preferably in a physiologically compatible buffer such as Hink solution, Ringer solution or physiological saline buffer. The solution may contain pharmaceutical agents (formulating agents) such as suspending, stabilizing and/or dispersing agents. Alternatively, the β -hairpin peptidomimetics of the invention may be in powder form for mixing with a suitable vehicle, e.g., sterile pyrogen-free water, prior to use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation, as is known in the art.
For oral administration, these compounds can be readily formulated by mixing the active β -hairpin peptidomimetics of the invention with pharmaceutically acceptable carriers well known in the art. Such carriers allow the β -hairpin peptidomimetics of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral formulations, such as powders, capsules and tablets, suitable excipients include fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agent; and a binder. If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. If desired, the solid dosage form can be manufactured as a sugar-coated tablet or enteric-coated tablet using standard techniques.
For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, and the like. In addition, flavoring agents, preservatives, coloring agents and the like may be added.
For oral administration, the compositions may take the form of tablets, lozenges and the like, which are generally formulated.
For administration by inhalation, the β -hairpin peptidomimetics of the invention may be conveniently delivered from a pressurized pack or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas, conveniently in the form of an aerosol spray. For pressurized aerosols, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, e.g., gels, containing a powder mixture of the β -hairpin peptidomimetics of the invention with a suitable powder base such as lactose or starch may be formulated for use in an inhaler or insufflator.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories, with suitable suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described above, the β -hairpin peptidomimetics of the invention can also be formulated as depot preparations (depot preparations). Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For the preparation of such depot preparations, the β -hairpin peptidomimetics of the invention can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble salts.
In addition, other drug delivery systems may be used, such as liposomes and emulsions known in the art. Certain organic solvents such as dimethyl sulfoxide may also be used. In addition, the β -hairpin peptidomimetics of the invention can be delivered by using a sustained release system, such as a semipermeable solid polymer matrix containing the therapeutic agent. Various sustained release materials have been established and are well known to those skilled in the art. Sustained release capsules can, depending on their chemical nature, release the compound over a period of several weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic agent, additional strategies for protein stabilization may be used.
Because the β -hairpin peptidomimetics of the invention may contain charged residues, they may be included in any of the above formulations as such or in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free form.
The β -hairpin peptidomimetics of the invention, or their compositions, are generally used in an amount effective to achieve the intended purpose. It will be appreciated that the amount will depend on the particular application.
For topical administration to treat or prevent HIV infection, a therapeutically effective dose can be determined by using in vitro assays such as those provided in the examples. The treatment may be used when the HIV infection is visible, or even when it is not visible. One of ordinary skill in the art will be able to determine, without undue experimentation, a therapeutically effective amount for treating a topical HIV infection.
For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays. For example, the dose can be formulated in animal models to achieve a circulating β -hairpin peptidomimetic concentration range that includes the IC as determined in cell culture50(i.e., the concentration of test compound that causes 50% death of the cell culture). This information can be used to more accurately determine useful doses in humans.
Initial dosages can also be determined from in vivo data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize dosing in humans based on animal data.
The dosage administered as an anti-HIV agent can be adjusted on the individual to provide plasma levels of the β -hairpin peptidomimetics of the invention sufficient to maintain the therapeutic effect. Therapeutically effective serum levels can be achieved by administering multiple doses per day.
For local administration or selective uptake, the effective local concentration of the β -hairpin peptidomimetics of the invention may not be related to plasma concentrations. One of ordinary skill in the art will be able to optimize therapeutically effective dosages for topical administration without undue experimentation.
The amount of β -hairpin peptidomimetic administered will, of course, depend on the subject to be treated, the weight of the subject, the severity of the patient, the mode of administration, and the judgment of the prescribing physician.
anti-HIV treatment may be repeated intermittently, when HIV infection is detectable or even when infection is not detectable. The treatment may be provided alone or in combination with other drugs, such as other anti-HIV agents or anti-cancer agents or other anti-microbial agents.
Generally, therapeutically effective doses of the beta-hairpin peptidomimetics described herein will provide therapeutic effects without causing substantial toxicity.
Toxicity of the beta-hairpin peptidomimetics of the invention can be determined by standard pharmaceutical methods in cell cultures or experimental animals, for example by determining the LD50(dose leading to 50% mortality in the population) or LD100(the dose that caused 100% mortality in the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate non-toxic dosage ranges for human use. The dosage of the beta-hairpin peptidomimetics of the invention is preferably in the range of severalWithin a circulating concentration range of effective doses that are not considered toxic or at all toxic. The dosage may vary within a certain range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by the individual physician taking into account the condition of the patient (see, e.g., Fingl et al 1975 in the pharmacological Basis of Therapeutics, Ch.1, p.1).
The following examples illustrate the invention in more detail but are not intended to limit the scope of the invention in any way. The following abbreviations are used in these examples:
HBTU: 1-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (Knorr et al Tetrahedron Lett.1989, 30, 1927-;
HOBt: 1-hydroxybenzotriazole;
DIEA: diisopropylethylamine;
HOAT: 7-aza-1-hydroxybenzotriazole;
HATU: o- (7-aza-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (Carpino et al, Tetrahedron Lett.1994, 35, 2279-.
Examples
1. Peptide synthesis
Coupling of the first protected amino acid residue to the resin
0.5g of 2-chlorotrityl chloride resin (Barlos et al, Tetrahedron Lett.1989, 30, 3943-. Suspending the resin in CH2Cl2(2.5ml) and allowed to swell at room temperature for 30 minutes under constant stirring. The resin was treated with 0.415mMol (leq) of the first suitably protected amino acid residue (see below) and 284. mu.l (4eq) of Diisopropylethylamine (DIEA) in CH2Cl2(2.5ml) and the mixture was shaken at 25 ℃ for 4 hours. Color of resinIt turned purple and the solution remained pale yellow. Resin was oscillated (CH)2Cl2MeOH/DIEA: 17/2/1), 30ml for 30 minutes; then using CH in the following order2Cl2(1×)，DMF(1×)，CH2Cl2(1X), methanol (1X), CH2Cl2(1X), methanol (1X), CH2Cl2(2×)，Et2Washed O (2 ×) and then dried under vacuum for 6 hours.
The loading is typically 0.6-0.7 mMol/g.
The following pre-loaded resins were prepared: Fmoc-ProO-chlorotrityl resin, Fmoc-DproO-chlorotrityl resin, and Fmoc-S- (4-S-Alloc-amino) -ProO-chlorotrityl resin.
Synthesis of fully protected peptide fragments
The synthesis was performed by using 24 to 96 reaction vessels with a Syro-peptide synthesizer (Multisyntech). To each vessel was added 60mg (weight of resin before loading) of the above resin. The following reaction cycle was programmed and run as follows:
step reagent time
1CH2Cl2Washed and swollen (by hand) for 3X 1min.
2DMF, washing and swelling for 1X 5min
340% piperidine/DMF 1X 5min.
4DMF, 5X 2min washing.
55 equivalents Fmoc amino acid/DMF
+5eq.HBTU
+5eq.HOBt
+5eq.DI EA 1×120min.
6DMF, 4X 2min washing.
7CH2Cl2Wash (at the end of synthesis) 3 × 2min repeat steps 3 to 6 to add each amino acid.
Pendant amino functionality Peg with 2- [2- (2-methoxyethoxy) ethoxy ] acetic acid and 2- (2-methoxyethoxy) acetic acid
The peptide-containing resin (0.040mmol) was added to 5ml of freshly distilled CH2Cl2Middle swelling for 30 min, then adding Pd (PPh) as a palladium catalyst3)414mg, 0.3 eq, then PhSiH is added30.8mmol, 20 equivalents. The resin was shaken for 2 hours, and the reaction solution was filtered off. The reaction was repeated again by using the same amount of reagent, after 2 hours the resin was CH-coated2Cl2And DMF and finally Et2And O washing.
The resin is again in freshly distilled CH2Cl2(2ml) for 30 minutes, the solvent was filtered off and the resin was swollen in DMF for 1 hour. A solution of DIPEA (10 equivalents) in 1ml of DMF was added followed by the addition of 2- [2- (2-methoxyethoxy) ethoxy]Acetic acid or 2- (2-methoxyethoxy) acetic acid (5 eq), and finally a solution of HATU (5 eq) in 1ml of DMF was added. The resin was shaken for 3 hours, and the reaction solution was filtered off. The reaction was repeated again by using the same amount of reagent, after 3 hours the resin was CH-coated2Cl2And DMF and finally Et2And O washing.
After the synthesis of the fully protected peptide fragment has been terminated, a pegylation procedure is optionally performed, then followed by procedure a, procedure B or procedure C as described below, depending on whether no interchain bonds are formed or β -chain disulfide bonds or β -chain lactam bonds are formed. Procedure a: cyclization and backbone cyclization peptide work-up
Fragmentation of fully protected peptide fragments
After completion of the synthesis, the resin was suspended in 1ml (0.39mMol) of CH2Cl21% TFA (v/v) for 3 min, filtered and the filtrate was diluted with 1ml (1.17mMol, 3 equiv) of aqueous solution in CH2Cl2DIEA (v/v) of 20% in (1). This procedure was repeated twice to ensure completion of the cleavage reaction. The filtrate was evaporated to dryness and the product was completely deprotected [ cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% Triisopropylsilane (TIS)]By reverse phase-HPLC (column C)18) And ESI-MS analysis to monitor the efficiency of the linear peptide synthesis.
The linear peptide is cyclized
100mg of fully protected linear peptide was dissolved in DMF (9ml, concentration 10 mg/ml). 41.8mg (0.110mMol, 3 equiv.) of HATU, 14.9mg (0.110mMol, 3 equiv.) of HOAt and 1ml (0.584mMol) of 10% DIEA in DMF (v/v) were then added and the mixture was vortexed at 20 ℃ for 16 h, then concentrated under high vacuum. The residue is in CH2Cl2And H2O/CH3And CN (90/10: v/v). Evaporating the CH2Cl2Phase, a fully protected cyclic peptide is obtained.
Deprotection and purification of cyclic peptides
The cyclic peptide obtained was dissolved in 1ml of a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% Triisopropylsilane (TIS). The mixture was allowed to stand at 20 ℃ for 2.5 hours and then concentrated under vacuum. The residue is dissolved in H2O/acetic acid (75/25: v/v), and the mixture was extracted with diisopropyl ether.
The aqueous phase is dried under vacuum and the product is then purified by preparative reverse phase HPLC.
The product was obtained as a white powder after lyophilization and then analyzed by ESI-MS. Analytical data including purity after preparative HPLC and ESI-MS are shown in tables 1, 2 and 3.
Analytical method 1:
analytical HPLC residence time (RT, min) by using VYDAC 218MS5215 column with the following solvent a (H)2O + 0.02% TFA) and B (CH)3CN) and gradient to determine: and (3) 0 minute: 92% A, 8% B; 8 minutes: 62% A38% B; 9-12 minutes: 0% A, 100% B.
Analysis method 2:
analytical HPLC residence time (RT, min) by using EX (s.n.217808-2) column with the following solvent a (H)2O + 0.02% TFA) and B (CH)3CN) and gradient to determine: and (3) 0 minute: 95% of A, 5% of B; 8 minutes: 30% A70% B; 9 minutes: 0% a, 100% B; 9-12 minutes: 95% of A and 5% of B. Procedure B: cyclization and work-up of backbone cyclized peptides with disulfide beta-chain linkages
Disulfide beta-chain bond formation
After completion of the synthesis, the resin was swollen in 3ml of dry DMF for 1 hour. Then 10 equivalents of iodine solution in DMF (6ml) were added to the reactor, followed by stirring for 1.5 hours. The resin was filtered and a fresh solution of iodine (10 equivalents) in DMF (6ml) was added, followed by stirring for another 3 hours. The resin was filtered with DMF (3X) and CH2Cl2(3X) washing.
Backbone cyclization, fragmentation and purification of peptides
After disulfide beta-chain bond formation, the resin was suspended in 1ml (0.39mMol) of a solution in CH2Cl21% TFA (v/v) for 3 min, filtered, and the filtrate diluted with 1ml (1.17mMol, 3 equiv) of aqueous solution in CH2Cl220% DIEA (v/v) in (C) was used for neutralization. This procedure was repeated twice to ensure completion of the fracture.
Volatiles were removed and 6ml of dry DMF was added to the tube. Then 2 equivalents of HATU in dry DMF (1ml) and 4 equivalents of DIPEA in dry DMF (1ml) were added to the peptide, followed by stirring for 16 hours. The volatiles were evaporated to dryness. The crude cyclized peptide was dissolved in 7ml of CH2Cl2In H2O (4.5ml)Extracted three times with 10% acetonitrile. CH (CH)2Cl2The layer was evaporated to dryness. To completely deprotect the peptide, 3ml of a cleaved cocktail (cocktail) TFA: TIS: H was added2O (95: 2.5), and the mixture was held for 2.5 hours. The volatiles were evaporated to dryness and the crude peptide was dissolved in 20% AcOH in water (7ml) and extracted three times with diisopropyl ether (4 ml). The aqueous layer was collected and evaporated to dryness and the residue was purified by preparative reverse phase HPLC.
The product was obtained as a white powder after lyophilization and then analyzed by ESI-MS analytical method 1 or 2. Analytical data including purity after preparative HPLC and ESI-MS are shown in tables 1, 2 and 3.
Procedure C: cyclisation and work-up of backbone cyclised peptides with lactam beta-chain bond
Formation of lactam beta-chain bonds
0.036mmol of resin was added to the reactor and swollen in dry DMF for 1 hour. 41.60mg (1 equivalent) of Pd (PPH) was added thereto3)4And 0.133ml (30 equiv.) of PhSiH3And then stirred overnight. The resin was filtered and washed thoroughly with DCM and DMF. The resin was again swollen in dry DMF for 1 hour. To this was added 1ml of DIPEA solution in DMF (24.64 μ L of DIPEA in 1ml of DMF, 4 equivalents), followed by 1ml of HATU solution in DMF (27.37mg of HATU, 2 equivalents), the final volume of the reaction mixture was 7ml, then stirred overnight. DMF, CH for resin2Cl2，DMF，CH2Cl2The washing was thorough.
Backbone cyclization, fragmentation and purification of peptides
The peptide was cleaved from the resin using 1% TFA in DCM, evaporated to dryness and 8ml of dry DMF was added to the tube. 2 equivalents of HATU in dry DMF (1ml) and 4 equivalents of DIPEA in dry DMF (1ml) were added to the peptide followed by stirring for 16 h. The volatiles were evaporated to dryness. The crude cyclized peptide was dissolved in 7ml of DCM and used in H210% acetonitrile in O (4.5ml) was extracted three times. Evaporate DCM layer to dryness.
The crude cyclized peptide was dissolved in 7ml of CH2Cl2In H210% acetonitrile in O (4.5ml) was extracted three times. CH (CH)2Cl2The layer is evaporated to dryness. To completely deprotect the peptide, 3ml of a cleaved cocktail (cocktail) TFA: TIS: H was added2O (95: 2.5), and the mixture was held for 2.5 hours. The volatiles were evaporated to dryness and the crude peptide was dissolved in 20% acetic acid in water (7ml) and extracted three times with diisopropyl ether (4 ml). The aqueous layer was collected and evaporated to dryness and the residue was purified by preparative reverse phase HPLC.
The product was obtained as a white powder after lyophilization and then analyzed by ESI-MS analytical method 1 or 2. Analytical data including purity after preparative HPLC and ESI-MS are shown in tables 1, 2 and 3.
Examples 1-6 and 8-11 (n-12) are shown in table 1. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above, in the following order: resin-Pro-DPro-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, they were cleaved from the resin, cyclized, deprotected and purified as shown in procedure a.
HPLC-retention time (min) was determined by using the gradient method 1 as described above:
Ex.1(4.98)；Ex.2(4.62)；Ex.3(5.63)；Ex.4(5.33)；Ex.5(5.12)，Ex.6(4.75)；Ex.8(5.08)；Ex.9(6.17)；Ex.10(6.28)；Ex.11(6.57)。
wherein "ex." means "examples".
Examples 7 and 12-14 (n-12) are shown in table 1. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, the peptide was cleaved from the resin, cyclized, deprotected and purified as shown in procedure B, forming disulfide bonds.
HPLC-retention time (min) was determined by using the gradient method 1 as described above:
Ex.7(4.48)；Ex.12(4.83)；Ex.13(5.30)；Ex.14(4.08)。
examples 15-50(n ═ 14) are shown in table 2. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, they were cleaved from the resin, cyclized, deprotected and purified as shown in procedure a.
HPLC-retention time (min) was determined by using the gradient method 1 as described above:
Ex.15(5.35)；Ex.16(5.48)；Ex.17(5.85)；Ex.18(5.78)；Ex.19(4.82)；Ex.20(5.33)；Ex.21(5.77)，Ex.22(5.85)；Ex.23(6.22)；Ex.24(6.22)；Ex.25(4.48)；Ex.26(5.08)；Ex.27(6.17)；Ex.28(6.28)；Ex.29(6.57)；Ex.30(6.73)；Ex.31(5.60)；Ex.32(5.58)；Ex.33(5.85)，Ex.34(6.20)；Ex.35(6.33)；Ex.36(5.43)；Ex.37(5.85)；Ex.38(5.92)；Ex.39(5.47)；Ex.40(6.0，6.37)*；Ex.41(5.13)；Ex.42(5.00)；Ex.43(5.00)；Ex.44(5.33，5.67)*，Ex.45(5.03)；Ex.46(4.75)；Ex.47(5.27)；Ex.48(5.65，6.08)*；Ex.49(5.03)；Ex.50(5.75)。
doublets showing correct MS.
Examples 51-115, 117-141, 143-148 (n-14) are shown in table 2. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
The HPLC-residence times (min) were determined by using the gradient method 1 (for examples Ex 51-53, 138-:
Ex.51(4.68)；Ex.52(4.67)；Ex.53(5.05)，Ex.54(3.16)，Ex.55(3.41)，Ex.56(3.07)，Ex.57(2.95)，Ex.58(2.99)，Ex.59(3.18)，Ex.60(3.16)，Ex.61(3.27)，Ex.62(2.91)，Ex.63(2.88)，Ex.64(2.88)，Ex.65(2.98)，Ex.66(3.17)，Ex.67(2.93)，Ex.68(2.91)，Ex.69(2.90)，Ex.70(2.88)，Ex.71(3.08)，Ex.72(3.00)，Ex.73(3.14)，Ex.74(3.02)，Ex.75(2.99)，Ex.76(3.56)，Ex.77(3.14)，Ex.78(3.18)，Ex.79(3.02)，Ex.80(3.18)，Ex.81(3.13)，Ex.82(3.38)，Ex.83(3.27)，Ex.84(3.32)，Ex.85(3.37)，Ex.86(3.57)，Ex.87(3.35)，Ex.88(3.08)，Ex.89(3.10)，Ex.90(3.14)，Ex.91(3.18)，Ex.92(3.17)，Ex.93(3.25)，Ex.94(3.10)，Ex.95(3.18)，Ex.96(3.15)，Ex.97(3.31)，Ex.98(3.26)，Ex.99(3.32)，Ex.100(3.28)，Ex.101(3.83)，Ex.102(3.00)，Ex.103(3.29)，Ex.104(2.98)，Ex.105(2.77)，Ex.106(2.74)，Ex.107(3.00)，Ex.108(2.81)，Ex.109(2.69，2.75)，Ex.110(2.76，2.82*)，Ex.111(2.73，2.78)，Ex.112(2.71)，Ex.113(2.51)，Ex.114(2.97)，Ex.115(2.95)，Ex.117(2.70)，Ex.118(2.78)，Ex.119(2.83)，Ex.120(2.80)，Ex.121(3.09)，Ex.122(3.45)，Ex.123(2.82)，Ex.124(3.29)，Ex.125(3.27)，Ex.126(3.19)，Ex.127(3.05)，Ex.128(3.86)，Ex.129(4.76)，Ex.130(4.43)，Ex.131(4.57)，Ex.132(4.45)，Ex.133(4.39)，Ex.134(4.27)，Ex.135(4.33)，Ex.136(2.75)，Ex.137(2.72)，Ex.138(4.75)，Ex.139(4.25)，Ex.140(4.77)，Ex.141(3.27)，Ex.143(3.01)，Ex.144(3.24)，Ex.145(2.84)，Ex.146(2.80)，Ex.147(2.91)，Ex.148(2.76)。
doublets showing correct MS.
Example 116(n ═ 14) is shown in table 2. From grafting to resinThe amino acid Pro above starts to synthesize the peptide. The starting resin was Fmoc-S- (4-S-Alloc-amino) -ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-S- (4-S-Alloc-amino) Pro-DPro-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Then 2- [2- (2-methoxyethoxy) ethoxy]Acetic acid to perform the pegylation procedure to give a templateDProA 8' 42. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.116 (3.00).
Example 142 (n-14) is shown in table 2. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P14-P13-P12-P11-P10-P9-Dab-P7-P6-P5-P4-P3-P2-P1. Then 2- [2- (2-methoxyethoxy) ethoxy]Acetic acid to perform the pegylation procedure. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.142 (3.18).
Example 149(n ═ 14) is shown in table 2. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DGln-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.149 (2.76).
Example 150(n ═ 14) is shown in table 2. From amino acids grafted onto resinsDPro begins to synthesize the peptide. The starting resin was Fmoc-DProO-chlorotrityl resin, which is prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-DPro-Gly-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, disulfide bridges are formed, the peptide cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.150 (2.61).
Example 151(n ═ 14) is shown in table 2. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, the peptide was cleaved from the resin, cyclized, deprotected and purified as shown in procedure a.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.151 (2.86).
Example 152-. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-DPro-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, a lactam bond is formed, the peptide cleaved from the resin, cyclized, deprotected and purified as shown in procedure C.
HPLC-retention time (min) was determined by using gradient method 2 as described above:
Ex.152(2.87)，Ex.153(2.87，2.88*)。
doublets showing correct MS.
Example 154-. From amino acids grafted onto resinsDPro begins to synthesize the peptide. The starting resin was Fmoc-DProO-chlorotrityl resin, which is prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-DPro-Gly-P18-P17-P16-P15-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Disulfide bonds were thereafter formed by using the following procedure:
to form a disulfide bond at positions P4 and P17, the protected cyclic peptide (36. mu. mol) was swollen in dry DMF for 1 hour. The DMF was taken off and replaced by 2ml of NMP and 444. mu.l of tri-n-butylphosphine (50 equivalents) under argon. The resin was shaken for 2 hours. The solvent was removed and the resin was washed once with 5ml NMP. Thereafter, the resin was again shaken with 2ml of NMP and 444. mu.l of tri-n-butylphosphine (50 equivalents) under argon for 2 hours. The resin was washed with DMF and transferred with 90ml DMF to a 250ml flask. 1mmol (330mg) of [ K ] in 10ml of water3Fe(CN)6]Added to the suspension and stirred gently overnight in the dark at 25 ℃. The resin was transferred to the reactor and washed once with 7ml water and twice with 5ml DMF.
To form a second disulfide bond at positions P8 and P13, the peptide was treated with 9 equivalents (83mg) of iodine in 6ml of dry DMF for 2 hours. The resin was washed once with DMF and the treatment with 9 equivalents (83mg) of iodine in 6ml of DMF was repeated. The resin was washed three times with 5ml DMF followed by 5ml CH2Cl2Washed three times. Thereafter, the peptide was cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.154(3.18), ex.155 (3.06).
Purity: % purity of compound after preparative HPLC: ex.154(97), ex.155 (95).
Quality: [ M +3H ]/3: ex.154(785.4), ex.155 (875.4).
Example 156-. From amino acids grafted onto resinsDPro begins to synthesize the peptide. The starting resin was Fmoc-DProO-chlorotrityl resin, which is prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-DPro-Gly-P18-P17-P16-P15-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.156(3.00), ex.157 (2.98).
Purity: % purity of compound after preparative HPLC: ex.156(95), ex.157 (76).
Quality: [ M +3H ]/3: ex.156(845.5), ex.157 (848.8).
Example 158 and 159(n- -18) are shown in Table 3. The peptide was synthesized starting from the amino acid Pro grafted onto the resin. The starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-Pro-Gly-P18-P17-P16-P15-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Thereafter, disulfide bonds are formed, the peptide is cleaved from the resin, cyclized, deprotected and purified as shown in procedure B.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.158(3.41), Ex.159(3.25)
Purity: % purity of compound after preparative HPLC: ex.158(95), Ex.159(83)
Quality: [ M +3H ]/3: ex.158(848.8), ex.159 (822.1).
Example 160(n ═ 18) is shownIn table 3. From amino acids grafted onto resinsDPro begins to synthesize the peptide. The starting resin was Fmoc-DproO-chlorotrityl resin, prepared as described above. The linear peptide was synthesized on the solid support according to the procedure described above in the following order: resin-DPro-Gly-P18-P17-P16-P15-P14-P13-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. The following procedure was used thereafter:
the peptide (36. mu. mol) was used in CH2Cl2The 1% TFA in (E) was cleaved from the resin. After evaporation to dryness, 8ml of dry DMF was added to the tube. Then 2 equivalents of HATU in dry DMF (1ml) and 4 equivalents of DIPEA in dry DMF (1ml) were added followed by stirring for 16 h. The volatiles were evaporated to dryness. The crude cyclized peptide was dissolved in 7ml of DCM and used in H210% acetonitrile in O (4.5ml) was extracted three times. The organic layer was evaporated to dryness. To completely deprotect the peptide, 3ml of a cleaved cocktail (cocktail) TFA: TIS: H was added2O (95: 2.5), the mixture was held for 3 hours. The volatiles were evaporated to dryness and the crude peptide was dissolved in 20% acetic acid in water (7ml) and extracted three times with diisopropyl ether (4 ml). The aqueous layer was diluted to 200ml with water. The pH was adjusted to a pH of 8 with ammonium hydroxide solution. The reaction mixture was shaken for 24 hours. The solution was acidified to pH5 with acetic acid, evaporated to dryness and purified by HPLC.
HPLC-retention time (min) was determined by using gradient method 2 as described above: ex.160(2.92)
Purity: % purity of compound after preparative HPLC: ex.160(93)
Quality: [ M +3H ]/3: ex.160 (785.3).
2. Biological method
2.1. Preparation of peptides
Unless otherwise stated, the lyophilized peptides were weighed on a microbalance (Mettler MT5) and dissolved in sterile water to a final concentration of 1 mM. The stock solution was kept at +4 ℃ in the dark.
2.2.Ca2+And (3) analysis: CXCR 4-antagonistic activity of the peptide.
The method comprises the following steps: Pre-B cells transfected with 3-4Mio CXCR4 were measured each time [ see references 1, 2 and 3 below]Resuspend in 200. mu.L MSB (20mM4- (2-hydroxyethyl) -piperazine-1-ethanesulfonic acid (HEPES), 136mM NaCl, 4.8mM KCl and 1mM CaCl) containing 5mM D-glucose2) In (1) and loaded with 0.75. mu.l of 1mM Fura-2-acetoxymethyl ester (Fura-2-AM) for 17 minutes at 37 ℃. The cells were centrifuged with plateletsFura-2-AM was washed off by machine and resuspended in 800. mu.L MSB containing 5mM D-glucose. The peptide to be administered was diluted 100-fold in MSB/0.2% PPL to the final concentration and 8. mu.L was injected. [ Ca ] in response to a single or sequential stimulation of a peptide2+]iDependent fluorescence changes were recorded with a fluorimeter at an excitation wavelength of 340nM and an emission wavelength of 510nM [ see reference 4 below ]]. The measurement was carried out at 37 ℃ with continuous stirring. The signal intensity was measured with 3mM CaCl2Per 1mM ionomycin (maximum fura-2-acetoxymethyl ester saturation) and 10. mu.M MnCl2Corrected for (minimum Fura-2-acetoxymethyl ester saturation) [ Ca2+]iChanges are given as% fura-2-acetoxymethyl ester saturation. [ Ca ]2+]iThe rate of change is at the initial [ Ca ]2+]iCalculating on the basis of the variation and plotting the curve as a function of the concentration of the chemokine to obtain a sigmoidal curve and determine the IC50The value is obtained.
MSB：20mM HEPES，136mM NaCl，4.8mM KCl，1mM CaCl2·2H2O, pH7.4; osmolarity (Osmolarity): adjusted with NaOH or HCl, with H2O or PBS regulated 310 mOsm.
MSB +: 5mM D-glucose in MSB (50mg/50 mL).
fura 2-acetoxymethyl ester: stock solution 1mM in dimethyl sulfoxide.
The method 2 comprises the following steps: the increase in intracellular calcium was monitored by using Flexstation 384(molecular devices, Sunnyvale, CA) to analyze CXCR4 antagonism of the peptide in mouse pre-B cell lines 300-19 that had been stably transfected with human CXCR4 [ see references 1, 2 and 3 below]. Cells were loaded in Assay buffer (Hanks balanced salt solution, HBSS, 20mM HEPES, ph7.4, 0.1% BSA) in batches with Calcium 3 Assay kit (Molecular Devices) for 1 hour at room temperature, and then 200,000 labeled cells were dispensed into 96-well black Assay plates (Costar No. 3603). A 20-fold concentrated solution of the peptide in assay buffer was added to these cells and the entire plate was centrifuged to settle the cells to the bottom of the well.Calcium mobilization induced by 10nM matrix-derived factor-1 (SDF-1) was measured in Flexstation 384 (challenge, 485 nM; emission, 525nM) for 90 seconds. The maximum change in fluorescence response above baseline was used to calculate antagonist activity. Data for dose-response curves (antagonist concentration versus% maximal response) four-parameter logistic equation was fitted using SoftmaxPro 4.6(molecular devices) from which the IC was calculated50% value.
2.3.FIGS-AssayTM
The analysis was performed according to reference 5 below. Stock dilutions (10mM) of the peptides were prepared by dissolving in 10mM Tris-HCl at room temperature. The stock solution was kept at +4 ℃ in the dark. Working dilutions were prepared by temporally serial dilutions in Phosphate Buffered Saline (PBS) and added directly to the cell culture in a final volume of 10 μ Ι. After 48 hours of co-cultivation the cultures were rinsed with PBS and then exposed to glutaraldehyde/formaldehyde (0.2%/2%) in PBS for five minutes. For photometric quantitative analysis, the immobilized cultures were subsequently incubated with o-nitro-phenyl-galactopyranoside (ONPG) as β -galactosidase substrate, which was enzymatically converted into the chromophore o-nitrophenol (ONP). This reading can be obtained directly by measuring the optical density of the wells at 405nm in an iEMS 96-well plate reader.
2.4. Cytotoxicity assays
Cytotoxicity of the peptide against HELA cells (Acc57) and COS-7 cells (CRL-1651) was determined by using MTT reduction assay [ see references 6 and 7 below]. Briefly, the method is as follows: HELA cells and COS-7 cells were cultured at 7.0. multidot.103Or 4.5.103Cells/well were seeded and incubated at 37 ℃ in 5% CO2Growth was performed in 96-well microtiter plates for 24 hours. At this point, the time zero (Tz) is determined by MTT reduction (see below). The supernatant of the remaining wells was removed and fresh medium and 12.5, 25 and 50 μ M serial dilutions of peptide were pipetted into each well. Each peptide concentration was analyzed in triplicate. Cell culture in 5% CO2Middle at 37The reaction was continued at ℃ for 48 hours. The wells were then washed once with PBS, and then 100. mu.l of MTT reagent (0.5 mg/mL in medium RPMI1640 and DMEM, respectively) was added to the wells. Then incubated at 37 ℃ for 2 hours, after which the medium was aspirated (aspirated) and 100. mu.l of isopropanol was added to each well. The absorbance (OD) at 595nm of the dissolved product was measured595Peptides). For each concentration, the average was calculated from triplicate experiments. The percentage of growth was calculated as follows: (OD)595peptide-OD595Tz-OD595Empty well)/(OD595Tz-OD595Empty wells) x 100%, a curve is plotted for each peptide concentration.
For each peptide, the LC 50 value (lethal concentration, defined as the concentration lethal to 50% of the cells) was determined by using the trendline function of EXCEL for the concentrations (50, 25, 12.5 and 0 μ M), the corresponding growth percentages and the value-50, (═ TREND (C50: C0,% 50: 0, -50)).
For each peptide, by targeting the concentration (50, 25, 12.5 and 0 μ g/ml), the corresponding percentage of growth and the value-50, (═ TREND (C)50∶C0，％50∶％050) calculating the GI 50 (growth inhibition) concentration using the trend line function.
2.5. Cell culture
"CCR 5" cells were cultured in DMEM medium supplemented with 50U/ml penicillin and 50. mu.g/ml streptomycin (Pen/Strept.) with 4500mg/ml glucose, 10% Fetal Bovine Serum (FBS). Hut/4-3 cells were maintained in RPMI medium, supplemented Pen/strept, and 10mM HEPES and 10% FBS. HELA cells and CCRF-CEM cells were maintained in RPMI1640 plus 5% FBS, Pen/Strept and 2mM L-glutamine. Cos-7 cells were grown in DMEM medium supplemented with 10% FCS, Pen/strept, and 2mM L-glutamine and having 4500mg/ml glucose. All cell lines were 5% CO2Medium at 37 ℃. Cell culture medium, medium supplement, PBS-buffer, HEPES, Pen/strept, L-glutamine and serum were purchased from Gibco (Pailsey, uk). All fine chemicals were from MERCK (Darmstadt, Germany).
2.6. Haemolysis
The peptide was tested for hemolytic activity against human red blood cells (hRBC). Fresh hRBCs were treated by centrifugation at 2000 Xg for 10 minutes and washed three times with Phosphate Buffered Saline (PBS). Peptides at a concentration of 100. mu.M were incubated with 20% v/v hRBC for 1 hour at 37 ℃. The final red blood cell concentration was about 0.9X 109Cells per ml. In the presence of PBS alone or in H2hRBC were incubated in the presence of 0.1% TritonX-100 in O to determine 0% and 100% cytolysis values, respectively. The samples were centrifuged, the supernatant diluted 20-fold in PBS buffer, and the Optical Density (OD) of the samples was measured at 540 nM. 100% lysis value (OD)540H20) An OD of about 1.3 to 1.8 is obtained540. The percent hemolysis was calculated as follows: (OD)540peptide/OD540H20)×100％。
2.7. Chemotaxis assay (cell migration assay)
Chemotactic response of CCRF-CEM cells to a gradient of stromal cell derived factor 1 alpha (SDF-1) was determined by using Neuroprobe disposable assay plates (5 μ pore size) (Gaithersburg, Md.) according to the manufacturer's instructions and references therein [ especially reference 8 below ]]To be measured. Briefly, one 175cm2The flask was washed once with Dubecco Phosphate Buffered Saline (DPBS), trypsinized for 10 minutes or until the cells had risen (lifted). Trypsin was neutralized by adding fresh medium containing serum, then cells were pelleted, washed once in DPBS and washed at 1-0.5X 107Individual cells/ml were resuspended in RPMI + 0.5% Bovine Serum Albumin (BSA). Mu.l of the cell suspension was mixed with 5. mu.l of 10-fold concentrated PEM peptide diluted in the same assay medium. 35. mu.l of this mixture were applied to the top of the analysis filter. Cells were allowed to migrate (at 37 ℃) into the lower chamber of the assay plate containing 1nM SDF-1. After 4 hours, the filter was removed, MTT was added to the migrated cells to a final concentration of 0.5mg/ml, and incubated for another 4 hours. After labeling with MTT, all media were removed and 100. mu.l of isopropanol +10mM HCl was addedInto these cells. Reading of light absorption at 595nm (ABS) by use of a Tecan Genios plate reader with Magellan software595). By combining ABS595The values were compared to standard curves generated in assay plates with known numbers of cells to determine the number of migrated cells and plotted against SDF-1 concentration to obtain sigmoidal curves and determine IC50The value is obtained. IC was determined by fitting averaged data points to a logarithmic curve using the trendline function in Microsoft Excel50The value is obtained.
2.8 plasma stability
mu.L of plasma/albumin solution was added to a polypropylene (PP) tube and spiked with 45. mu.l of compound from 100. mu.M solution B obtained from 15. mu.1 mM peptide in PBS (pH7.4) and 135. mu.l PBS. A150 μ L aliquot was transferred to each well of a10 kDa filter plate (Millipore MAPPB 1010 Biomax membrane). For the "0 min control": add 270. mu.L of PBS to the PP tube and add 30. mu.L of stock solution B, vortex. 150 μ L of control solution was added to one well of the filter plate and served as "filtered control".
In addition, another 150. mu.l of the control solution was added directly to the receiving well (retained to the filtrate) and served as "unfiltered control". The entire plate including the evaporation lid was incubated at 37 ℃ for 60 minutes. Plasma samples (mouse plasma: Harlan Sera lab UK, human plasma: Blutspendeszentrum Surich) were centrifuged at 4300rpm (3500g) at 15 ℃ for at least 2 hours to give 100. mu.L of filtrate. For "serum albumin" samples (freshly prepared human albumin: Sigma A-4327, mouse albumin: Sigma A-6272, all at a concentration of 40mg/ml in PBS), a centrifugation treatment of about 1 hour was sufficient. The filtrate in the receiving PP plates was analyzed by LC/MS as follows: column: jupiter C18(Phenomenex), mobile phase: (A) 0.1% formic acid and (B) acetonitrile in water, gradient: 5% -100% in 2min (B), electrospray ionization, MRM detection (triple quadrupole). Peak areas were determined and triplicate assay values were averaged. The binding was performed as follows (filtered and unfiltered time points 0 min)Expressed as a percentage of controls 1 and 2: 100- (100 × T)60/T0). The average of these values is then calculated (see reference 9 below).
2.9. Pharmacokinetic studies (PK)
Pharmacokinetic studies after a single intravenous (i.v.) and intraperitoneal (i.p.) administration were performed for the compound of example 51 ("Ex 51"). 30g (+ 20%) male CD-1 mice obtained from Charles river laboratories Deutschland GmbH were used in this study. Vehicle saline was added to achieve a final concentration of 1mg/ml of compound. The volumes were 2ml/kg i.v. and 10ml/kg i.p., and peptide ex.51 was injected to achieve a final intraperitoneal dose of 10mg/kg and an intravenous dose of 2 mg/kg. Approximately 250 and 300 μ l of blood were drawn from the retroorbital plexus under mild isoflurane anesthesia and added to heparinized tubes at predetermined time intervals (0, 5, 15, 30 minutes and 1, 2 and 3 hours for the i.v. study and 0, 15, 30 minutes and 1, 2, 4 and 8 hours for the i.p. study). Plasma was separated from the precipitated cells by centrifugation and frozen at-80 ℃ before use for HPLC-MS analysis.
Preparation of plasma calibration samples
"blank" mouse plasma from untreated animals was used. 50ng of propranol (internal standard, IS) was spiked into 0.2ml aliquots of plasma (by solid phase extraction in OASIS)_Sample preparation was performed on HLB bridges (Waters) and a known amount of ex.51 was spiked in order to obtain 9 plasma calibration samples in the range 10-5000 nM. OASIS_The HLB column was conditioned with 1ml of methanol, followed by 1ml of 1% NH3And (5) conditioning the aqueous solution. The sample was then incubated with 700. mu.l of 1% NH3The aqueous solution was diluted and loaded. Plates were plated with 1ml of methanol/1% NH3The aqueous solution 5/95 is washed. Elution was performed using 1ml of 0.1% TFA in methanol.
The plates containing the eluate were introduced into a concentrator system and dried. The residue was dissolved in 100. mu.l of formic acid 0.1%/acetonitrile 95/5(v/v) and analyzed in HPLC/MS on a reverse phase analytical column (Jupiter C18, 50X 2.0mm, 5 μm, Phenomenex) using a gradient elution (mobile phase A: 0.1% aqueous formic acid, B: acetonitrile; from 5% B to 100% B, over 2 minutes).
Preparation of plasma samples
Samples from animal treatments were pooled in order to obtain a suitable volume for extraction. If the total volume obtained is less than 0.2ml, a suitable amount of "blank" mouse plasma is added in order to maintain the same matrix as the calibration curve. The samples were then doped with IS and processed as described for the calibration curve.
Pharmacokinetic evaluation
By using the software PK solutions 2.0TM(Summit Research Service, Montrose, CO 81401USA) PK analyses were performed on pooled data (typically n 2 or 3). The area under the curve AUC is calculated by the linear trapezoidal rule. AUC(t-∞)Estimated as Ct/b (b: elimination rate constant). AUC(t-∞)Is AUC(0-t)And AUC(t-∞)The sum of (a) and (b). The elimination half-life is calculated by linear regression on at least three data points in the elimination phase. The time interval chosen for half-life determination is determined by the correlation coefficient (r)2) It is evaluated that the correlation coefficient should be at least higher than 0.85 and optimally higher than 0.96. For the case of i.v. administration, at tzeroThe lower starting concentration was determined by extrapolating the curve through the first two time points. Finally bioavailability after i.p. administration is by i.p. versus normalized AUC after i.v. administration(0-∞)And (4) calculating the ratio.
3.0. Results
The results of the experiments described under 2.2 to 2.7 above are shown in table 4 below.
TABLE 4
n.d.: not determined
Ca for Ex.1-532+IC in assay50The (nM) values were determined by using method 1, and for Ex.54-155, method 2 was used. For determination of cytotoxicity values, LC was used in Ex.1-5350Calculation method, for Ex.52-160, GI was used50And (4) calculating.
Table 4 (continuation)
n.d. not determined
Ca for Ex.1-532+IC in assay50The (nM) values were determined by using method 1, and for Ex.54-160, method 2.
The results of the experiment described under 2.8 above are shown in table 5 below.
TABLE 5
Ex. Stable human plasma t1/2(min) Stable rat plasma t1/2(min)
51 286 ＞300
60 ＞300 ＞300
61 273 ＞300
68 127 81
75 188 142
85 166 ＞300
101 ＞300 247
102 255 245
110 115 259
124 ＞300 ＞＞300
120 39 174
151 89 71
152 23 86
The results of the experiment described in 2.9(PK) above are shown in tables 6, 7 and 8 below.
TABLE 6
Pathway(s) i.v.
Dosage form 2mg/kg
Time (h.) Calculated concentration (ng/ml) Number of animals pooled
0.083 1461 3
0.25 328 2
0.5 300 3
1 80 3
2 68 3
3 49 3
TABLE 7
Pathway(s) i.p.
Dosage form 10mg/kg
Time (h.) Calculated concentration (ng/ml) Number of animals pooled
0.25 673 3
0.5 1568 2
1 2009 2
2 3160 2
4 1024 3
8 519 3
TABLE 8
Route of administration Intravenous administration of drugs Intraperitoneal cavity
Dose (mg/kg) AUC0-t(ng·h/ml)AUC0-inf.(ng.h/ml)AUCnorm.(ng.h/ml)Cmax ng/mlCmax norm.TmaxBeta (hour) of-1) Half-life (hours)% absorption (F) (normalized AUC0-inf.Intraperitoneal vs normalized AUC0-infPercentage of i.v.) 217041905953285941429700.242.8100 1011112129481295316031620.282.5136
The intravenous kinetics of ex.51 were followed after intravenous administration of ex.51 at a dose level of 2mg/kg body weight. Ex.51 showed an extrapolated C of 28594ng/ml after PK analysisinitialAnd 1461ng/ml observed C at 5minmax. Plasma levels rapidly decreased to 328 and 80ng/ml at 15 minutes or 1 hour, respectively. From 1 to 3 hours, plasma levels were reduced with an elimination half-life of 2.8 hours to 49ng/ml at 3 hours. AUC(0-t)And AUC(0-∞)1704 and 1905ng.h/ml were reached, respectively.
After intraperitoneal administration of Ex.51 at a dose level of 10mg/kg body weight, the plasma level of Ex.51 increased almost linearly over the first 2 hours and showed 3160ng/ml of C at 2 hoursmax. From 2 to 8 hours, plasma levels were reduced with an elimination half-life of 2.5 hours to 519ng/ml at 8 hours. AUC(0-t)And AUC(0-∞)11112 and 12948ng.h/ml are respectively reached. I.v. dosing with ex.51 (100% absorption,953ng.h/ml) compared to absorption of 136% (1295ng.h/ml) after i.p. administration and a normalized Cmax 45-fold lower after i.p. administration (316 vs 1497 ng/ml). Values above 100% may reflect, in part, the reduction in reliability due to the limited number of data points.
Reference to the literature
1.Oberlin E，Amara A，Bachelerie F，Bessia C，Virelizier J-L，Arenzana-Seisdedos F，Schwartz O，Heard J-M，Clark-Lewis I，Legler DF，Loetscher M，Baggiolini M，Moser B.Nature.1996，382：833-835
2.Loetscher M，Geiser T，O’Reilly T，Zwalen R，Baggiolini M，Moser B.J.Biol.Chem.1994.269：232-237
3.D’Apuuo M，Rolink A，Loetscher M，Hoxie JA，Clark-Lewis I，Melchors F，Baggiolini M，Moser B.Eur.J.Immunol.1997.27：1788-1793
4.Von Tscharner V，Prod′hom B，Baggiolini M，Reuter H.Nature.1986.324：369-72.
5.Hamy F，Felder ER，Heizmann G，Lazdins J，Aboul-ela F，VaraniG，Karn J，Klimkait T.Proc.Natl.Acad.Sci.1997.94：3548-3553.
6.Mossman T.J.Immunol.Meth.1983，65：55-63
7.Berridge MV，Tan AS.Arch.Biochm.Biophys.1993，303：474-482
8.Frevert CW，Wong VA，Goodman RV，Goodwin R，Martin TR，J.Immunol.Meth.1998.213：41-52
9.Singh R.，Chang，S.Y.，Talor，L.C.，Rapid Commun.MassSpectrom.，1996，10：1019-1026

Claims (37)

1. A compound of the general formula and its pharmaceutically acceptable salts,
wherein
Is thatDPro-LPro；
Z is a chain of N α -amino acid residues, N being an integer 12 or 14, the positions of said amino acid residues in said chain being counted starting from the N-terminal amino acid, whereby these amino acid residues are, depending on their position in the chain:
if n is 12, the α -amino acid residues at positions 1 to 12 in the chain Z are:
-P1：Tyr；
-P2：Arg，Gly；
-P3：Cit；
-P4：Val，Phe，Gly，Ile，Thr，Gln，Cys；
-P5：Arg；
-P6：Arg，DArg；
-P7：Arg；
-P8：Trp，2-Nal；
-P9：Val，Phe，Gly，Ile，Thr，Gln，Cys；
-P10：Tyr；
-P11: cit, Gly; and
-P12: lys; or
-disulfide bond formation at P4 with Cys at P9; or
If n is 14, the α -amino acid residues at positions 1 to 14 are:
-P1：Tyr，Gln，Arg，His，Ile，Trp，Thr，Glu，Ser，Val，Met，Phe，Gly，Asp，Leu；
-P2：Arg，His，Lys，4-PyrAla；
-P3：Cit；Arg，His，Ile，Tyr，Trp，Pro，Glu，Asn，Asp，Lys，Ala，Leu，Val，4F-Phe，Met，Ser，Thr，Gln，Tyr；
-P4：Val，Phe，Tyr，t-BuG，Cys，Ser，Dab，Glu；
-P5：Arg，Dab，Ser，(EA)G；
-P6：Pro，Gly，Phe，Val，Cit，Ala；
-P7：DPro，Pro，Gly，Val；
-P8：Arg，Tyr，Trp，Thr，4F-Phe，Dab，4-PyrAla，Isorn，(Im)G，Cit，His，IpegDab；
-P9：Arg，(Pip)G，(EA)G，Orn；
-P10：2-Nal，Trp，Tyr；
-P11：Phe，Tyr，Val，t-BuG，Cys，Asn，Glu，Dab；
-P12：Tyr，Cit；
-P13: cit, Gln, Arg, His, Tyr, Asn, Asp, Lys, Ala, Ser, Leu, Met, NMeGly, Thr; and
-P14: lys, Glu, Gln, Asn, Asp, Ala, Ser, NMeK; or
-disulfide bond formation at P4 with Cys at P11; or
-Glu at P4 forming a lactam bond with Dab at P11; or
-Dab at P4 forms a lactam bond with Glu at P11;
provided that
-the amino acid residue at position P1 is Gly; and/or
-the amino acid residue at position P3 is Glu, Asn, Asp, Thr, or gin; and/or
-the amino acid residue at position P4 is Cys, Ser or Glu; and/or
-the amino acid residue at position P5 is Ser or (EA) G; and/or
-the amino acid residue at position P6 is Phe, Val or Ala; and/or
-the amino acid residue at position P7 is Val, Pro, orDPro; and/or
-the amino acid residue at position P8 is Tyr, Trp, 4F-Phe, 4-PyrAla, (Im) G, His orDPro; and/or
-the amino acid residue at position P9 is (EA) G; and/or
-the amino acid residue at position P11 is Val or t-BuG; and/or
-the amino acid residue at position P12 is Tyr or Cit; and/or
-the amino acid residue at position P13 is Glu, gin, Asp, Asn, Ser, Thr, Cys or NMeGly; and/or
-disulfide bond formation at P4 with Cys at P11; and/or
-Glu at P4 forming a lactam bond with Dab at P11; and/or
Dab at P4 forming a lactam bond with Glu at P11.
2. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Phe；
-P5：Arg；
-P6：Val；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Val；
-P12：Tyr；
-P13：Cit；
-P14：Lys。
3. the compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Phe；
-P5：Arg；
-P6：Val；
-P7：Pro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Val；
-P12：Tyr
-P13：Cit；
-P14：Lys。
4. the compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Phe；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Phe；
-P12：Tyr
-P13：Cit；
-P14：Lys。
5. the compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Val；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Val；
-P12：Tyr；
-P13：Cit；
-P14：Lys。
6. the compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Cit；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
7. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：Gly；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Cit；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
8. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Va1；
-P7：Gly；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Cit；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
9. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Trp；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
10. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Thr；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
11. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Gln；
disulfide bonds are formed between P4 and Cys at P11.
12. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Glu；
disulfide bonds are formed between P4 and Cys at P11.
13. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and in position 1-the amino acid residues at 14 are:
-P1：Glu；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
14. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Glu；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
15. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Asn；
disulfide bonds are formed between P4 and Cys at P11.
16. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Asp；
disulfide bonds are formed between P4 and Cys at P11.
17. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Ser；
disulfide bonds are formed between P4 and Cys at P11.
18. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Ser；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
19. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Ser；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：Trp；
-P11：Cys；
-P12：Tyr
-P13：Cit；
-P14：Asp；
disulfide bonds are formed between P4 and Cys at P11.
20. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：His；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：Trp；
-P11：Cys；
-P12：Tyr
-P13：Cit；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
21. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Thr；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Dab；
-P9：Arg；
-P10：Trp；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
22. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：His；
-P3：Thr；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：His；
-P10：Trp；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
23. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：2-Nal；
-P11：Cys；
-P12：Tyr
-P13：Thr；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
24. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Ile；
-P4：Cys；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：Tyr；
-P11：Cys；
-P12：Tyr
-P13：Gln；
-P14：Lys；
disulfide bonds are formed between P4 and Cys at P11.
25. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Ser；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：Trp；
-P11：Asn；
-P12：Tyr
-P13：Cit；
-P14：Lys；
dab at P4 forms a lactam bond with Glu at P11.
26. The compound of formula I according to claim 1, wherein the template isDPro-LPro, n is 14 and the amino acid residues at positions 1-14 are:
-P1：Tyr；
-P2：Arg；
-P3：Cit；
-P4：Dab；
-P5：Arg；
-P6：Gly；
-P7：DPro；
-P8：Arg；
-P9：Arg；
-P10：Trp；
-P11：Glu；
-P12：Tyr
-P13：Cit；
-P14：Lys；
the Glu at position P11 forms a lactam bond with Dab at position P4.
27. A pharmaceutical composition comprising a compound according to any one of claims 1 to 26 and a pharmaceutically inert carrier.
28. A composition according to claim 27, which is in a form suitable for oral, topical, transdermal, injection, buccal, transmucosal, pulmonary or inhalation administration.
29. A composition according to claim 27 or 28, which is in the form of a tablet, dragee, capsule, liquid, gel, paste, suspension, spray, or suppository.
30. A composition according to claim 27 or 28, which is in the form of a solution or a slurry.
31. The composition of claim 27 or 28, which is in the form of a cream, ointment, syrup, or nebulizer.
32. Use of a compound according to any one of claims 1 to 26 for the manufacture of a CXCR4 antagonist drug.
33. The use of claim 32, wherein said CXCR4 antagonist drug is intended for use in preventing HIV infection in healthy individuals or slowing and stopping viral development in infected patients; or when cancer is mediated or caused by CXCR4 receptor activity; or when immunological diseases are mediated or caused by CXCR4 receptor activity; or for the treatment of immunosuppression; or used in the reinjection collection process of the peripheral blood stem cells.
34. A process for the manufacture of a compound according to any one of claims 1 to 26, which process comprises:
(a) coupling a suitably functionalized solid support with a suitably N-protected derivative of an amino acid at position 5, 6 or 7 in the desired end product if N is 12 or at position 6, 7 or 8 in the desired end product if N is 14, any functional groups that may be present in the N-protected amino acid derivative also being suitably protected;
(b) removing the N-protecting group from the product obtained;
(c) coupling the product obtained with a suitably N-protected derivative of an amino acid located in the desired end product at a position closer to the N-terminal amino acid residue, any functional groups that may be present in the N-protected amino acid derivative being likewise suitably protected;
(d) removing the N-protecting group from the product obtained;
(e) repeating steps (c) and (d) until the N-terminal amino acid residue is introduced;
(f) allowing the product obtained to stand
(fa) withLCoupling of an appropriately N-protected derivative of Pro;
(fb) removing the N-protecting group from the obtained product; and
(fc) reacting the product obtained withDCoupling of an appropriately N-protected derivative of Pro;
(g) removing the N-protecting group from the product obtained in step (fc);
(h) coupling the product obtained with a suitably N-protected derivative of an amino acid which is located at position 12 if N is 12 or at position 14 if N is 14 in the desired end product, any functional groups which may be present in said N-protected amino acid derivative being likewise suitably protected;
(i) removing the N-protecting group from the product obtained;
(j) coupling the product obtained with a suitably N-protected derivative of an amino acid which is located at a position further away from position 12 in the desired end product if N is 12 or at a position further away from position 14 if N is 14, any functional group which may be present in said N-protected amino acid derivative being likewise suitably protected;
(k) removing the N-protecting group from the product obtained;
(1) repeating steps (j) and (k) until all amino acid residues are introduced;
(m) optionally, selectively deprotecting one or more protected functional groups present in the molecule and appropriately replacing the released reactive groups;
(n) optionally, one, two or three interchain bonds are formed between the side chains of suitable amino acid residues located at opposite positions of the β -strand region;
(o) detaching the obtained product from the solid support;
(p) cyclizing the product cleaved from the solid support;
(q) removing, optionally, any protecting groups present on the functional groups of any members of the chain of amino acid residues, any protecting groups that may otherwise be present in the molecule;
(r) optionally, converting the obtained product into a pharmaceutically acceptable salt or converting the obtained pharmaceutically acceptable or unacceptable salt into the corresponding free compound of formula I or into a different pharmaceutically acceptable salt.
35. A process for the manufacture of a compound according to any one of claims 1 to 26, which process comprises:
(a') letting an appropriately functionalized solid support
(a' a) withLCoupling of an appropriately N-protected derivative of Pro;
(a' b) removing the N-protecting group from the obtained product; and
(a' c) reacting the product obtained withDCoupling of an appropriately N-protected derivative of Pro;
(b ') removing the N-protecting group from the product obtained in step (a' c);
(c') coupling the product obtained with a suitably N-protected derivative of an amino acid which is in position 12 if N is 12 or in position 14 if N is 14 in the desired end product, any functional groups which may be present in said N-protected amino acid derivative being likewise suitably protected;
(d') removing the N-protecting group from the product obtained;
(e') coupling the product obtained with a suitably N-protected derivative of an amino acid which is located at a position further away from position 12 in the desired end product if N is 12 or at a position further away from position 14 if N is 14, any functional group which may be present in said N-protected amino acid derivative also being suitably protected;
(f') removing the N-protecting group from the product obtained;
(g ') repeating steps (e ') and (f ') until all amino acid residues have been introduced;
(h') optionally, selectively deprotecting one or more protected functional groups present in the molecule and appropriately replacing the released reactive groups;
(i') optionally, forming one, two or three interchain bonds between side chains of suitable amino acid residues located at opposite positions of the β -strand region;
(j') detaching the product obtained from the solid support;
(k') cyclizing the product cleaved from the solid support;
(l') removing, optionally, any protecting groups present on the functional groups of any member of the chain of amino acid residues, any protecting groups that may otherwise be present in the molecule; and
(m') optionally, converting the obtained product into a pharmaceutically acceptable salt or converting the obtained pharmaceutically acceptable or unacceptable salt into the corresponding free compound of formula I or into a different pharmaceutically acceptable salt.
36. A process according to claim 34 or 35, but wherein the residue of an N-substituted glycine residue is introduced by coupling with an acylating agent containing a leaving group followed by nucleophilic displacement with a primary amine corresponding to the desired substituent, wherein optionally the amine is suitably protected.
37. The process according to claim 36 wherein said acylating agent containing a leaving group is bromo, chloro or iodoacetic acid.
HK06114147.7A 2003-05-02 2004-04-29 Template-fixed beta-hairpin peptidomimetics with cxcr4 antagonizing activity HK1094210B (en)

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PCT/EP2003/004640 WO2004096839A1 (en) 2003-05-02 2003-05-02 Template-fixed beta-hairpin peptidomimetics with cxcr4 antagonizing activity
EPPCT/EP03/04640 2003-05-02
PCT/EP2004/004535 WO2004096840A1 (en) 2003-05-02 2004-04-29 Template- fixed beta-hairpin peptidomimetics with cxcr4 antagonizing activity

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HK1094210A1 HK1094210A1 (en) 2007-03-23
HK1094210B true HK1094210B (en) 2012-09-07

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